Nucleic acids and corresponding proteins entitled 282p1g3 useful in treatment and detection of cancer

ABSTRACT

A novel gene 282P1G3 and its encoded protein, and variants thereof, are described wherein 282P1G3 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 282P1G3 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 282P1G3 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 282P1G3 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of United States non-provisional utility patent application U.S. Ser. No. 12/400,679, filed 9 Mar. 2009, which is a continuation of United States non-provisional utility patent application U.S. Ser. No. 11/438,944, filed 23 May 2006, now U.S. Pat. No. 7,612,172, issued 3 Nov. 2009, which is a continuation of U.S. Ser. No. 10/435,751, filed 9 May 2003, now U.S. Pat. No. 7,115,727, issued 3 Oct. 2006. This application claims priority from United States provisional patent application U.S. Ser. No. 60/404,306, filed 16 Aug. 2002, and from United States provisional patent application U.S. Ser. No. 60/423,290, filed 1 Nov. 2002. The contents of the applications listed in this paragraph are fully incorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to genes and their encoded proteins, termed 282P1G3, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 282P1G3.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 511582008403SeqList.txt, date recorded: Jun. 7, 2013, size: 1,012).

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 282P1G3, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 282P1G3 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 282P1G3 are provided. The tissue-related profile of 282P1G3 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 282P1G3 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary to all or part of the 282P1G3 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 282P1G3-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 282P1G3-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 282P1G3 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 282P1G3 genes, mRNAs, or to 282P1G3-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 282P1G3. Recombinant DNA molecules containing 282P1G3 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 282P1G3 gene products are also provided. The invention further provides antibodies that bind to 282P1G3 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 282P1G3 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 282P1G3. A typical embodiment of this invention provides methods for monitoring 282P1G3 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 282P1G3 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 282P1G3 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 282P1G3 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 282P1G3. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 282P1G3 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 282P1G3 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 282P1G3 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 282P1G3. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 282P1G3 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 282P1G3 production) or a ribozyme effective to lyse 282P1G3 mRNA.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables VIII-XXI and XXII to XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 282P1G3 SSH sequence of 321 nucleotides.

FIG. 2. A) The cDNA and amino acid sequence of 282P1G3 variant 1 (also called “282P1G3 v.1” or “282P1G3 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 272-3946 including the stop codon.

B) The cDNA and amino acid sequence of 282P1G3 variant 2 (also called “282P1G3 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3787 including the stop codon.

C) The cDNA and amino acid sequence of 282P1G3 variant 3 (also called “282P1G3 v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-2953 including the stop codon.

D) The cDNA and amino acid sequence of 282P1G3 variant 4 (also called “282P1G3 v.4”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3625 including the stop codon.

E) The cDNA and amino acid sequence of 282P1G3 variant 5 (also called “282P1G3 v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3898 including the stop codon.

F) The cDNA and amino acid sequence of 282P1G3 variant 6 (also called “282P1G3 v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3823 including the stop codon.

G) The cDNA and amino acid sequence of 282P1G3 variant 7 (also called “282P1G3 v.7”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3982 including the stop codon.

H) The cDNA and amino acid sequence of 282P1G3 variant 8 (also called “282P1G3 v.8”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3859 including the stop codon.

I) The cDNA and amino acid sequence of 282P1G3 variant 28 (also called “282P1G3 v.28”) is shown in FIG. 2I. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 192-3866 including the stop codon.

J) The cDNA and amino acid sequence of 282P1G3 variant 14 (also called “282P1G3 v.14”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3946 including the stop codon.

K) SNP variants of 282P1G3 v.1. 282P1G3 v.9 through v.25. The 282P1G3 v.9 through v.23 proteins have 1224 amino acids. Variants 282P1G3 v.9 through v.25 are variants with single nucleotide difference from 282P1G3 v.1. 282P1G3 v.9, v.10, v.11, v.24 and v.25 proteins differ from 282P1G3 v.1 by one amino acid. 282P1G3 v.12 through v.23, v.26 and v.27 code for the same protein as v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above in FIGS. 2A through 2I.

FIG. 3. A) The amino acid sequence of 282P1G3 v.1 is shown in FIG. 3A; it has 1224 amino acids.

B) The amino acid sequence of 282P1G3 v.2 is shown in FIG. 3B; it has 1171 amino acids.

C) The amino acid sequence of 282P1G3 v.3 is shown in FIG. 3C; it has 893 amino acids.

D) The amino acid sequence of 282P1G3 v.4 is shown in FIG. 3D; it has 1117 amino acids.

E) The amino acid sequence of 282P1G3 v.5 is shown in FIG. 3E; it has 1208 amino acids.

F) The amino acid sequence of 282P1G3 v.6 is shown in FIG. 3F; it has 1183 amino acids.

G) The amino acid sequence of 282P1G3 v.7 is shown in FIG. 3G; it has 1236 amino acids.

H) The amino acid sequence of 282P1G3 v.8 is shown in FIG. 3H; it has 1195 amino acids.

I) The amino acid sequence of 282P1G3 v.9 is shown in FIG. 3I; it has 1224 amino acids.

J) The amino acid sequence of 282P1G3 v.10 is shown in FIG. 3J; it has 1224 amino acids.

K) The amino acid sequence of 282P1G3 v.11 is shown in FIG. 3 k; it has 1224 amino acids.

L) The amino acid sequence of 282P1G3 v.24 is shown in FIG. 3L; it has 1224 amino acids.

M) The amino acid sequence of 282P1G3 v.25 is shown in FIG. 3M; it has 1224 amino acids.

As used herein, a reference to 282P1G3 includes all variants thereof, including those shown in FIGS. 2, 3, 10, and 11, unless the context clearly indicates otherwise.

FIG. 4. FIG. 4A: Alignment of 282P1G3 with human close homolog of L1 (gi 27894376). FIG. 4B: Alignment of 282P1G3 with mouse close homolog of L1 (gi6680936).

FIG. 5. FIGS. 5( a)-(c): Hydrophilicity amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 6. FIGS. 6( a)-(c): Hydropathicity amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 7. FIGS. 7( a)-(c): Percent accessible residues amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 8. FIGS. 8( a)-(c): Average flexibility amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 9. FIGS. 9( a)-(c): Beta-turn amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 10. Schematic alignment of SNP variants of 282P1G03 v.1. Variants 282P1G03 v.9 through v.27 are variants with single nucleotide difference from v.1. Variant v.14 inserted a ‘T’ between 4635 and 4636 of v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any transcript variants as shown in FIG. 12, e.g. v.2, that contains the bases. Numbers correspond to those of 282P1G03 v.1. Black box shows the same sequence as 282P1G03 v.1. SNPs are indicated above the box.

FIG. 11. Schematic alignment of protein variants of 282P1G03. Protein variants are named to correspond to nucleotide variants. Variants v.2 through v.8 were translated from splice variants. Variants v.7 and v.8 had an insertion of 12 amino acids. Variants v.9 through v.11, v.24, and v.25 were translated from SNP variants. Nucleotide variants 282P1G03 v.12 through v.23 coded for the same protein as v.1. Single amino acid differences among the proteins translated from SNP variants were indicated above the boxes. Black boxes represent the same sequence as 282P1G03 v.1. Numbers underneath the box correspond to positions in 282P1G03 v.1.

FIG. 12. Structures of transcript variants of 282P1G03. Variant 282P1G03 v.2 through v.8 and v.28 are transcript variants of 282P1G03 v.1. Variant 282P1G03 v.3 deleted exons 22 through 27, 3′ portion of exon 21 and 5′ portion of exon 28 of variant 282P1G03 v.1. Variants v.2, v.4, v.5 and v.6 spliced out exon 25, exons 21-22, exon 8, and exon 6, respectively, in v.1. Variant 282P1G03 v.7 extended 36 bp at the 5′ end of exon 11 of variant 282P1G03 v.1. In addition to such an extension of 36 bp to exon 11 of v.1, variant 282P1G03 v.8 deleted exon 6 of variant 282P1G03 v.1. The 11th potential exon had two forms: the longer form was 36 bp longer than the shorter form. The 21st and 28th potential exons could also have a long and a short form, as seen in v. 3. Poly A tails are not shown here. Numbers in “( )” underneath the boxes correspond to those of 282P1G03 v.1. Lengths of introns and exons are not proportional.

FIG. 13. Secondary structure and transmembrane domains prediction for 282P1G3B protein variants.

The secondary structure of 282P1G3B protein variants 1 through 8 (FIGS. 13A (SEQ ID NO: 199), 13B (SEQ ID NO: 200), 13C (SEQ ID NO: 201), 13D (SEQ ID NO: 202), 13E (SEQ ID NO: 203), 13F (SEQ ID NO: 204), 13G (SEQ ID NO: 205), and 13H (SEQ ID NO: 206) respectively) were predicted using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G., accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed.

FIGS. 13I, 13K, 13M, 13I, 13Q, 13S, 13U, and 13W: Show schematic representations of the probability of existence of transmembrane regions and orientation of 282P1G3B variants 1 through 9, respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS. 13J, 13L, 13N, 13P, 13R, 13T, 13V, and 13X: Show schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 282P1G3B variants 1 through 9, respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/).

FIG. 14. 282P1G3 Expression by RT-PCR. First strand cDNA was prepared from (A) vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool; (B) normal stomach, normal brain, normal heart, normal liver, normal skeletal muscle, normal testis, normal prostate, normal bladder, normal kidney, normal colon, normal lung, normal pancreas, and a pool of cancer specimens from pancreas cancer patients, ovary cancer patients, and cancer metastasis specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. (A) Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas. (B) Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression in pancreas cancer, ovary cancer, cancer metastasis, and normal brain compared to all other normal tissues tested.

FIG. 15. 282P1G3 expression in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/lane were probed with the 282P1G3 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 9-10 kb 282P1G3 transcript in normal brain, but not in any other normal tissue tested.

FIG. 16. Expression of 282P1G3 in Pancreas Cancer Patient Specimens. RNA was extracted from pancreas cancer cell lines (CL), normal pancreas (N), and pancreas cancer patient tumor (T). Northern blots with 10 ug of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in pancreas cancer patient tumor specimen but not in the cell lines nor in the normal pancreas.

FIG. 17. Expression of 282P1G3 in Ovary Cancer Patient Specimens. RNA was extracted from ovary cancer cell lines (CL), normal ovary (N), and ovary cancer patient tumor (T). Northern blots with 10 ug of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in ovary cancer patient tumor specimen but not in the cell lines nor in the normal ovary.

FIG. 18. Expression of 282P1G3 in Lymphoma Cancer Patient Specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with bug of total RNA were probed with the 282P1G3 sequence. Size standards in kilobases are on the side. Results show expression of 282P1G3 in lymphoma patient specimens but not in the normal blood cells tested.

FIG. 19. 282P1G3 Expression in 293T Cells Following Transfection of 282P1G3.pcDNA3.1/MycHis Construct. The complete ORF of 282P1G3 v.2 was cloned into the pcDNA3.1/MycHis construct to generate 282P1G3.pcDNA3.1/MycHis. 293T cells were transfected with either 282P1G3.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control. Forty hours later, cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression of 282P1G3 from the 282P1G3.pcDNA3.1/MycHis construct in the lysates of transfected cells.

FIG. 20. 282P1G3 Expression in 293T Cells Following Transfection of 282P1G3.pcDNA3.1/MycHis Construct. The extracellular domain, amino acids 26-1043, of 282P1G3 v.2 was cloned into the pTag5 construct to generate 282P1G3.pTag5. 293T cells were transfected with 282P1G3.pTag5 construct. Forty hours later, supernatant as well as cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression and secretion of 282P1G3 from the 282P1G3.pTag5 transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 282P1G3 Polynucleotides

II.A.) Uses of 282P1G3 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

II.A.2.) Antisense Embodiments

II.A.3.) Primers and Primer Pairs

II.A.4.) Isolation of 282P1G3-Encoding Nucleic Acid Molecules

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

III.) 282P1G3-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments     -   III.B.) Expression of 282P1G3-related Proteins     -   III.C.) Modifications of 282P1G3-related Proteins     -   III.D.) Uses of 282P1G3-related Proteins

IV.) 282P1G3 Antibodies

V.) 282P1G3 Cellular Immune Responses

VI.) 282P1G3 Transgenic Animals

VII.) Methods for the Detection of 282P1G3

VIII.) Methods for Monitoring the Status of 282P1G3-related Genes and Their Products

IX.) Identification of Molecules That Interact With 282P1G3

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines     -   X.B.) 282P1G3 as a Target for Antibody-Based Therapy     -   X.C.) 282P1G3 as a Target for Cellular Immune Responses     -   X.C.I. Minigene Vaccines     -   X.C.2. Combinations of CTL Peptides with Helper Peptides     -   X.C.3. Combinations of CTL Peptides with T Cell Priming Agents     -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or         HTL Peptides     -   X.D.) Adoptive Immunotherapy     -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic         Purposes

XI.) Diagnostic and Prognostic Embodiments of 282P1G3.

XII.) Inhibition of 282P1G3 Protein Function

-   -   XII.A.) Inhibition of 282P1G3 With Intracellular Antibodies     -   XII.B.) Inhibition of 282P1G3 with Recombinant Proteins     -   XII.C.) Inhibition of 282P1G3 Transcription or Translation     -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) Identification, Characterization and Use of Modulators of 282P1G3

XIV.) KITS/Articles of Manufacture

I.) Definitions

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 282P1G3 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 282P1G3. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 282P1G3-related protein). For example, an analog of a 282P1G3 protein can be specifically bound by an antibody or T cell that specifically binds to 282P1G3.

The term “antibody” is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-282P1G3 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-282P1G3 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-282P1G3 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”

A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi^(212 or 213), P³² and radioactive isotopes of Lu including Lu¹⁷⁷. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of FIG. 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of FIG. 2. In another embodiment, the sequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.

In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 282P1G3 genes or that encode polypeptides other than 282P1G3 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 282P1G3 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 282P1G3 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 282P1G3 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By “neutralize” is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein. The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.

The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 282P1G3-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.

The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in FIG. 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following (non-limiting exemplary uses are also set forth):

Examples of Medical Isotopes:

Isotope Description of use Actinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat (AC-227) metastases in the skeleton resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212) Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancer detection (Cd-109) Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators, and for (Co-60) sterilization of medical supplies Copper-64 A positron emitter used for cancer therapy and SPECT imaging (Cu-64) Copper-67 Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic (Cu-67) studies (i.e., breast and colon cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy (Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for the small joints associated (Er-169) with fingers and toes Europium-152 Radiation source for food irradiation and for sterilization of medical supplies (Eu-152) Europium-154 Radiation source for food irradiation and for sterilization of medical supplies (Eu-154) Gadolinium-153 Osteoporosis detection and nuclear medical quality assurance devices (Gd-153) Gold-198 Implant and intracavity therapy of ovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer (Ho-166) radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis treatment Iodine-125 Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer (I-125) treatment, radiolabeling, tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs Iodine-131 Thyroid function evaluation, thyroid disease detection, treatment of thyroid (I-131) cancer as well as other non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked (Ir-192) arteries (i.e., arteriosclerosis and restenosis), and implants for breast and prostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries (i.e., (Lu-177) arteriosclerosis and restenosis) Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, (Mo-99) liver, lungs, heart, and other organs. Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103 Prostate cancer treatment (Pd-103) Platinum-195m Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic (Pt-195m) drug Phosphorus-32 Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone (P-32) cancer diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and (P-33) treatment of blocked arteries (i.e., arteriosclerosis and restenosis) Radium-223 See Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and (Re-186) treatment of lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain (Re-188) relief, treatment of rheumatoid arthritis, and treatment of prostate cancer Rhodium-105 Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used in brain studies, imaging of adrenal cortex by gamma- (Se-75) scintigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool Strontium-85 Bone cancer detection and brain scans (Sr-85) Strontium-89 Bone cancer pain relief, multiple myeloma treatment, and osteoblastic (Sr-89) therapy Technetium-99m See Molybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer (Th-228) radioimmunotherapy Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) (Th-229) which are alpha emitters used in cancer radioimmunotherapy Thulium-170 Gamma source for blood irradiators, energy source for implanted medical (Tm-170) devices Tin-117m Cancer immunotherapy and bone cancer pain relief (Sn-117m) Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for cancer (W-188) diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis) Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary (Xe-127) function tests, and cerebral blood flow studies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer (Y-90) treatment Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer (Y-91) radioimmunotherapy (i.e., lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers)

By “randomized” or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequence preferences or constants at any position. In another embodiment, the library is a “biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind or interact with 282P1G3, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 282P1G3 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 282P1G3 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non-limiting constituents of various supertypes are as follows:

A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901, A*0207

A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101

B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701, B*7801, B*0702, B*5101, B*5602

B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)

A1: A*0102, A*2604, A*3601, A*4301, A*8001

A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003

B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08

B58: B*1516, B*1517, B*5701, B*5702, B58

B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 282P1G3 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.

The “282P1G3-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 282P1G3 proteins or fragments thereof, as well as fusion proteins of a 282P1G3 protein and a heterologous polypeptide are also included. Such 282P1G3 proteins are collectively referred to as the 282P1G3-related proteins, the proteins of the invention, or 282P1G3. The term “282P1G3-related protein” refers to a polypeptide fragment or a 282P1G3 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.

II.) 282P1G3 Polynucleotides

One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 282P1G3 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 282P1G3-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 282P1G3 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 282P1G3 gene, mRNA, or to a 282P1G3 encoding polynucleotide (collectively, “282P1G3 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.

Embodiments of a 282P1G3 polynucleotide include: a 282P1G3 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 282P1G3 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 282P1G3 nucleotides comprise, without limitation:

(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2, wherein T can also be U;

(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A, from nucleotide residue number 272 through nucleotide residue number 3946, including the stop codon, wherein T can also be U;

(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B, from nucleotide residue number 272 through nucleotide residue number 3787, including the stop codon, wherein T can also be U;

(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C, from nucleotide residue number 272 through nucleotide residue number 3953, including the a stop codon, wherein T can also be U;

(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D, from nucleotide residue number 272 through nucleotide residue number 3625, including the stop codon, wherein T can also be U;

(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2E, from nucleotide residue number 272 through nucleotide residue number 3898, including the stop codon, wherein T can also be U;

(VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F, from nucleotide residue number 272 through nucleotide residue number 3823, including the stop codon, wherein T can also be U;

(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G, from nucleotide residue number 272 through nucleotide residue number 3982, including the stop codon, wherein T can also be U;

(IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H, from nucleotide residue number 272 through nucleotide residue number 3859, including the stop codon, wherein T can also be U;

(X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2I, from nucleotide residue number 192 through nucleotide residue number 3866, including the stop codon, wherein T can also be U;

(XI) a polynucleotide that encodes a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-J;

(XII) a polynucleotide that encodes a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-J;

(XIII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;

(XIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XXXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XXXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XL) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XLI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XLII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XLIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XLIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XLV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XLVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XLVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XLVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(XLIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(L) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(LI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(LII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(LIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9

(LIV) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(LIII).

(LV) a peptide that is encoded by any of (I) to (LIV); and

(LVI) a composition comprising a polynucleotide of any of (I)-(LIV) or peptide of (LV) together with a pharmaceutical excipient and/or in a human unit dose form.

(LVII) a method of using a polynucleotide of any (I)-(LW) or peptide of (LV) or a composition of (LVI) in a method to modulate a cell expressing 282P1G3,

(LVIII) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3

(LIX) a method of using a polynucleotide of any (I)-(LW) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3, said cell from a cancer of a tissue listed in Table I;

(LX) a method of using a polynucleotide of any (I)-(LW) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat a a cancer;

(LXI) a method of using a polynucleotide of any (I)-(LW) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,

(LXII) a method of using a polynucleotide of any (I)-(LW) or peptide of (LV) or a composition of (LVI) in a method to identify or characterize a modulator of a cell expressing 282P1G3.

As used herein, a range is understood to disclose specifically all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 282P1G3 polynucleotides that encode specific portions of 282P1G3 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1210, 1220, and 1224 or more contiguous amino acids of 282P1G3 variant 1; the maximal lengths relevant for other variants are: variant 2, 1171 amino acids; variant 3, 893 amino acids, variant 4, 1117 amino acids, variant 5, 1208 amino acids, variant 6, 1183 amino acids, variant 7, 1236 amino acids, variant 8, 1195 amino acids, variant 9, 1224 amino acids, variant 10, 1224 amino acids, variant 11, 1224 amino acids, variant 24, 1224 amino acids, and variant 25, 1224 amino acids.

For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 282P1G3 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 282P1G3 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 282P1G3 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 282P1G3 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed herein include 282P1G3 polynucleotide fragments encoding one or more of the biological motifs contained within a 282P1G3 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 282P1G3 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 282P1G3 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 282P1G3 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

II.A.) Uses of 282P1G3 Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 282P1G3 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 282P1G3.” For example, because the 282P1G3 gene maps to this chromosome, polynucleotides that encode different regions of the 282P1G3 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 282P1G3 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 282P1G3 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 282P1G3 was shown to be highly expressed in prostate and other cancers, 282P1G3 polynucleotides are used in methods assessing the status of 282P1G3 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 282P1G3 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 282P1G3 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 282P1G3. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 282P1G3 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 282P1G3. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 282P1G3 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 282P1G3 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The 282P1G3 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 282P1G3 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 282P1G3 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 282P1G3 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 282P1G3 mRNA. Optionally, 282P1G3 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 282P1G3. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 282P1G3 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet. 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of these nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 282P1G3 polynucleotide in a sample and as a means for detecting a cell expressing a 282P1G3 protein.

Examples of such probes include polypeptides comprising all or part of the human 282P1G3 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 282P1G3 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 282P1G3 mRNA.

The 282P1G3 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 282P1G3 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 282P1G3 polypeptides; as tools for modulating or inhibiting the expression of the 282P1G3 gene(s) and/or translation of the 282P1G3 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 282P1G3 or 282P1G3 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 282P1G3-Encoding Nucleic Acid Molecules

The 282P1G3 cDNA sequences described herein enable the isolation of other polynucleotides encoding 282P1G3 gene product(s), as well as the isolation of polynucleotides encoding 282P1G3 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 282P1G3 gene product as well as polynucleotides that encode analogs of 282P1G3-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 282P1G3 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 282P1G3 gene cDNAs can be identified by probing with a labeled 282P1G3 cDNA or a fragment thereof. For example, in one embodiment, a 282P1G3 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 282P1G3 gene. A 282P1G3 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 282P1G3 DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containing a 282P1G3 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 282P1G3 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 282P1G3 or a fragment, analog or homolog thereof can be used to generate 282P1G3 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 282P1G3 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 282P1G3 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 282P1G3 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 282P1G3 and 282P1G3 mutations or analogs.

Recombinant human 282P1G3 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 282P1G3-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 282P1G3 or fragment, analog or homolog thereof, a 282P1G3-related protein is expressed in the 293T cells, and the recombinant 282P1G3 protein is isolated using standard purification methods (e.g., affinity purification using anti-282P1G3 antibodies). In another embodiment, a 282P1G3 coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 282P1G3 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 282P1G3 coding sequence can be used for the generation of a secreted form of recombinant 282P1G3 protein.

As discussed herein, redundancy in the genetic code permits variation in 282P1G3 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell. Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 282P1G3-Related Proteins

Another aspect of the present invention provides 282P1G3-related proteins. Specific embodiments of 282P1G3 proteins comprise a polypeptide having all or part of the amino acid sequence of human 282P1G3 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 282P1G3 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 282P1G3 shown in FIG. 2 or FIG. 3.

Embodiments of a 282P1G3 polypeptide include: a 282P1G3 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 282P1G3 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 282P1G3 peptides comprise, without limitation:

(I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-J or FIG. 3A-M;

(II) a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-J or 3A-M;

(III) a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-J or 3A-M;

(IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;

(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;

(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;

(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;

(VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;

(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M in any whole number increment up to 1224 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3A, 3I-3M in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3B in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3C in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3D in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXXI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXXII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXXIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3E in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXXIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXXV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXXVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXXVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXXVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3F in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXXIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XL) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XLI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XLII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XLIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3G in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XLIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XLV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XLVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XLVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XLVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3H in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XLIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;

(L) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;

(LI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;

(LII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;

(LIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;

(LW) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;

(LV) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;

(LVI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;

(LVII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide:

-   -   i) a region of at least 5 amino acids of a particular peptide of         FIG. 3, in any whole number increment up to the full length of         that protein in FIG. 3, that includes an amino acid position         having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,         or having a value equal to 1.0, in the Hydrophilicity profile of         FIG. 5;     -   ii) a region of at least 5 amino acids of a particular peptide         of FIG. 3, in any whole number increment up to the full length         of that protein in FIG. 3, that includes an amino acid position         having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or         having a value equal to 0.0, in the Hydropathicity profile of         FIG. 6;     -   iii) a region of at least 5 amino acids of a particular peptide         of FIG. 3, in any whole number increment up to the full length         of that protein in FIG. 3, that includes an amino acid position         having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,         or having a value equal to 1.0, in the Percent Accessible         Residues profile of FIG. 7;     -   iv) a region of at least 5 amino acids of a particular peptide         of FIG. 3, in any whole number increment up to the full length         of that protein in FIG. 3, that includes an amino acid position         having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,         or having a value equal to 1.0, in the Average Flexibility         profile of FIG. 8; or,     -   v) a region of at least 5 amino acids of a particular peptide of         FIG. 3, in any whole number increment up to the full length of         that protein in FIG. 3, that includes an amino acid position         having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,         or having a value equal to 1.0, in the Beta-turn profile of FIG.         9;

(LVIII) a composition comprising a peptide of (I)-(LVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.

(LIX) a method of using a peptide of (I)-(LVII), or an antibody or binding region thereof or a composition of (LVIII) in a method to modulate a cell expressing 282P1G3,

(LX) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3

(LXI) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition (LVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3, said cell from a cancer of a tissue listed in Table I;

(LXII) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;

(LXIII) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,

(LXIV) a method of using a a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition (LVIII) in a method to identify or characterize a modulator of a cell expressing 282P1G3.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 282P1G3 polynucleotides that encode specific portions of 282P1G3 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1210, 1215, 1220, and 1224 or more contiguous amino acids of 282P1G3 variant 1; the maximal lengths relevant for other variants are: variant 2, 1171 amino acids; variant 3, 893 amino acids, variant 4, 1117 amino acids, variant 5, 1208 amino acids, variant 6, 1183 amino acids, variant 7, 1236 amino acids, variant 8, 1195 amino acids, variant 9, 1224 amino acids, variant 10, 1224 amino acids, variant 11, 1224 amino acids, variant 24, 1224 amino acids, and variant 25, 1224 amino acids.

In general, naturally occurring allelic variants of human 282P 1G3 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 282P1G3 protein contain conservative amino acid substitutions within the 282P1G3 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 282P1G3. One class of 282P1G3 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 282P1G3 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2n^(d) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 282P1G3 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 282P1G3 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 282P1G3 variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 282P1G3 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 282P1G3 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 282P1G3 variant also specifically binds to a 282P1G3 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 282P1G3 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 282P1G3-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 282P1G3 protein variants or analogs comprises one or more of the 282P1G3 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 282P1G3 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 282P1G3 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 282P1G3 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 282P1G3 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 282P1G3 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

282P1G3-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 282P1G3-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 282P1G3 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed herein include 282P1G3 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 282P1G3 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., Epimatrix™ and Epimer™, Brown University, and BIMAS).

Motif bearing subsequences of all 282P1G3 variant proteins are set forth and identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfam searches on the INTERNET. The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.

Polypeptides comprising one or more of the 282P1G3 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 282P1G3 motifs discussed above are associated with growth dysregulation and because 282P1G3 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 282P1G3 protein that are capable of optimally binding to specified HLA alleles (e.g., Table W; Epimatrix™ and Epimer™, Brown University, and BIMAS). Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

282P1G3-related proteins are embodied in many forms, preferably in isolated form. A purified 282P1G3 protein molecule will be substantially free of other proteins or molecules that impair the binding of 282P1G3 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 282P1G3-related proteins include purified 282P1G3-related proteins and functional, soluble 282P1G3-related proteins. In one embodiment, a functional, soluble 282P1G3 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 282P1G3 proteins comprising biologically active fragments of a 282P1G3 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 282P1G3 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 282P1G3 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.

282P1G3-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-282P1G3 antibodies or T cells or in identifying cellular factors that bind to 282P1G3. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a 282P1G3 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at the World Wide Web; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, and BIMAS. Illustrating this, peptide epitopes from 282P1G3 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 282P1G3 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site on the World Wide Web; in addition to the site SYFPEITHI.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 282P1G3 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)) Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site for SYFPEITHI, or BIMAS) are to be “applied” to a 282P1G3 protein in accordance with the invention. As used in this context “applied” means that a 282P1G3 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 282P1G3 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 282P1G3-Related Proteins

In an embodiment described in the examples that follow, 282P1G3 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 282P1G3 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 282P1G3 protein in transfected cells. The secreted HIS-tagged 282P1G3 in the culture media can be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 282P1G3-Related Proteins

Modifications of 282P1G3-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 282P1G3 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 282P1G3 protein. Another type of covalent modification of a 282P1G3 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 282P1G3 comprises linking a 282P1G3 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 282P1G3-related proteins of the present invention can also be modified to form a chimeric molecule comprising 282P1G3 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 282P1G3 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 282P1G3. A chimeric molecule can comprise a fusion of a 282P1G3-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 282P1G3 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 282P1G3-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 282P1G3 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 282P1G3-Related Proteins

The proteins of the invention have a number of different specific uses. As 282P1G3 is highly expressed in prostate and other cancers, 282P1G3-related proteins are used in methods that assess the status of 282P1G3 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 282P1G3 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 282P1G3-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 282P1G3 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 282P1G3-related proteins that contain the amino acid residues of one or more of the biological motifs in a 282P1G3 protein are used to screen for factors that interact with that region of 282P1G3.

282P1G3 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 282P1G3 protein), for identifying agents or cellular factors that bind to 282P1G3 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 282P1G3 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 282P1G3 gene product. Antibodies raised against a 282P1G3 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 282P1G3 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 282P1G3-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 282P1G3 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 282P1G3-expressing cells (e.g., in radio scintigraphic imaging methods). 282P1G3 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 282P1G3 Antibodies

Another aspect of the invention provides antibodies that bind to 282P1G3-related proteins. Preferred antibodies specifically bind to a 282P1G3-related protein and do not bind (or bind weakly) to peptides or proteins that are not 282P1G3-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 282P1G3 can bind 282P1G3-related proteins such as the homologs or analogs thereof.

282P1G3 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 282P1G3 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 282P1G3 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 282P1G3 and mutant 282P1G3-related proteins. Such assays can comprise one or more 282P1G3 antibodies capable of recognizing and binding a 282P1G3-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 282P1G3 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 282P1G3 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 282P1G3 expressing cancers such as prostate cancer.

282P1G3 antibodies are also used in methods for purifying a 282P1G3-related protein and for isolating 282P1G3 homologues and related molecules. For example, a method of purifying a 282P1G3-related protein comprises incubating a 282P1G3 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 282P1G3-related protein under conditions that permit the 282P1G3 antibody to bind to the 282P1G3-related protein; washing the solid matrix to eliminate impurities; and eluting the 282P1G3-related protein from the coupled antibody. Other uses of 282P1G3 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 282P1G3 protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 282P1G3-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 282P1G3 can also be used, such as a 282P1G3 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 282P1G3-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 282P1G3-related protein or 282P1G3 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol 15: 617-648).

The amino acid sequence of a 282P1G3 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 282P1G3 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 282P1G3 amino acid sequence are used to identify hydrophilic regions in the 282P1G3 structure. Regions of a 282P1G3 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 282P1G3 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 282P1G3 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

282P1G3 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 282P1G3-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 282P1G3 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 282P1G3 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 282P1G3 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 282P1G3 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and, 6,114598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 282P1G3 antibodies with a 282P1G3-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 282P1G3-related proteins, 282P1G3-expressing cells or extracts thereof. A 282P1G3 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 282P1G3 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 282P1G3 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via the World Wide Web; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity, including:

1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including ⁵¹Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 282P1G3 Transgenic Animals

Nucleic acids that encode a 282P1G3-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 282P1G3 can be used to clone genomic DNA that encodes 282P1G3. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 282P1G3. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 issued 12 Apr. 1988, and 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 282P1G3 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 282P1G3 can be used to examine the effect of increased expression of DNA that encodes 282P1G3. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 282P1G3 can be used to construct a 282P1G3 “knock out” animal that has a defective or altered gene encoding 282P1G3 as a result of homologous recombination between the endogenous gene encoding 282P1G3 and altered genomic DNA encoding 282P1G3 introduced into an embryonic cell of the animal. For example, cDNA that encodes 282P1G3 can be used to clone genomic DNA encoding 282P1G3 in accordance with established techniques. A portion of the genomic DNA encoding 282P1G3 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 282P1G3 polypeptide.

VII.) Methods for the Detection of 282P1G3

Another aspect of the present invention relates to methods for detecting 282P1G3 polynucleotides and 282P1G3-related proteins, as well as methods for identifying a cell that expresses 282P1G3. The expression profile of 282P1G3 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 282P1G3 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 282P1G3 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of 282P1G3 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 282P1G3 polynucleotides include, for example, a 282P1G3 gene or fragment thereof, 282P1G3 mRNA, alternative splice variant 282P1G3 mRNAs, and recombinant DNA or RNA molecules that contain a 282P1G3 polynucleotide. A number of methods for amplifying and/or detecting the presence of 282P1G3 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 282P1G3 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 282P1G3 polynucleotides as sense and antisense primers to amplify 282P1G3 cDNAs therein; and detecting the presence of the amplified 282P1G3 cDNA. Optionally, the sequence of the amplified 282P1G3 cDNA can be determined.

In another embodiment, a method of detecting a 282P1G3 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 282P1G3 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 282P1G3 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 282P1G3 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a 282P1G3 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 282P1G3-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 282P1G3-related protein in a biological sample comprises first contacting the sample with a 282P1G3 antibody, a 282P1G3-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 282P1G3 antibody; and then detecting the binding of 282P1G3-related protein in the sample.

Methods for identifying a cell that expresses 282P1G3 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 282P1G3 gene comprises detecting the presence of 282P1G3 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 282P1G3 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 282P1G3, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 282P1G3 gene comprises detecting the presence of 282P1G3-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 282P1G3-related proteins and cells that express 282P1G3-related proteins.

282P1G3 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 282P1G3 gene expression. For example, 282P1G3 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 282P1G3 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 282P1G3 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 282P1G3-Related Genes and Their Products

Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 282P1G3 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 282P1G3 in a biological sample of interest can be compared, for example, to the status of 282P1G3 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 282P1G3 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Greyer et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 282P1G3 status in a sample.

The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 282P1G3 expressing cells) as well as the level, and biological activity of expressed gene products (such as 282P1G3 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 282P1G3 comprises a change in the location of 282P1G3 and/or 282P1G3 expressing cells and/or an increase in 282P1G3 mRNA and/or protein expression.

282P1G3 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 282P1G3 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 282P1G3 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 282P1G3 gene), Northern analysis and/or PCR analysis of 282P1G3 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 282P1G3 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 282P1G3 proteins and/or associations of 282P1G3 proteins with polypeptide binding partners). Detectable 282P1G3 polynucleotides include, for example, a 282P1G3 gene or fragment thereof, 282P1G3 mRNA, alternative splice variants, 282P1G3 mRNAs, and recombinant DNA or RNA molecules containing a 282P1G3 polynucleotide.

The expression profile of 282P1G3 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 282P1G3 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 282P1G3 status and diagnosing cancers that express 282P1G3, such as cancers of the tissues listed in Table I. For example, because 282P1G3 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 282P1G3 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 282P1G3 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 282P1G3 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 282P1G3 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 282P1G3 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 282P1G3 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 282P1G3 expressing cells (e.g. those that express 282P1G3 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 282P1G3-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 282P1G3 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 282P1G3 gene products by determining the status of 282P1G3 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 282P1G3 gene products in a corresponding normal sample. The presence of aberrant 282P1G3 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 282P1G3 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 282P1G3 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 282P1G3 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 282P1G3 mRNA or express it at lower levels.

In a related embodiment, 282P1G3 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 282P1G3 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 282P1G3 expressed in a corresponding normal sample. In one embodiment, the presence of 282P1G3 protein is evaluated, for example, using immunohistochemical methods. 282P1G3 antibodies or binding partners capable of detecting 282P1G3 protein expression are used in a variety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 282P1G3 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 282P1G3 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 282P1G3 indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 282P1G3 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 282P1G3 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prey., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of 282P1G3. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 282P1G3 expression. The presence of RT-PCR amplifiable 282P1G3 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 282P1G3 mRNA or 282P1G3 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 282P1G3 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 282P1G3 in prostate or other tissue is examined, with the presence of 282P1G3 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 282P1G3 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 282P1G3 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 282P1G3 mRNA or 282P1G3 protein expressed by tumor cells, comparing the level so determined to the level of 282P1G3 mRNA or 282P1G3 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 282P1G3 mRNA or 282P1G3 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 282P1G3 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 282P1G3 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 282P1G3 mRNA or 282P1G3 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 282P1G3 mRNA or 282P1G3 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 282P1G3 mRNA or 282P1G3 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 282P1G3 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 282P1G3 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., flocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between the expression of 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 gene products) and another factor associated with malignancy entails detecting the overexpression of 282P1G3 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 282P1G3 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 282P1G3 and PSA mRNA in prostate tissue is examined, where the coincidence of 282P1G3 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 282P1G3 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 282P1G3 mRNA include in situ hybridization using labeled 282P1G3 riboprobes, Northern blot and related techniques using 282P1G3 polynucleotide probes, RT-PCR analysis using primers specific for 282P1G3, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 282P1G3 mRNA expression. Any number of primers capable of amplifying 282P1G3 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 282P1G3 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 282P1G3

The 282P1G3 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 282P1G3, as well as pathways activated by 282P1G3 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999, 5,925,523 issued 20 Jul. 1999, 5,846,722 issued 8 Dec. 1998 and 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 282P1G3 protein sequences. In such methods, peptides that bind to 282P1G3 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 282P1G3 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 282P1G3 protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3 Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 282P1G3 are used to identify protein-protein interactions mediated by 282P1G3. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun 1999, 261:646-51). 282P1G3 protein can be immunoprecipitated from 282P1G3-expressing cell lines using anti-282P1G3 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 282P1G3 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, ³⁵5-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 282P1G3 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 282P1G3's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 282P1G3-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 282P1G3 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 282P1G3 function can be identified based on their ability to bind 282P1G3 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 282P1G3 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 282P1G3.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a 282P1G3 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 282P1G3 amino acid sequence, allowing the population of molecules and the 282P1G3 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 282P1G3 amino acid sequence, and then separating molecules that do not interact with the 282P1G3 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 282P1G3 amino acid sequence. The identified molecule can be used to modulate a function performed by 282P1G3. In a preferred embodiment, the 282P1G3 amino acid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 282P1G3 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.

For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.

Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., “cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.

Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.

Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.

Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.

Accordingly, therapeutic approaches that inhibit the activity of a 282P1G3 protein are useful for patients suffering from a cancer that expresses 282P1G3. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 282P1G3 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 282P1G3 gene or translation of 282P1G3 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 282P1G3-related protein or 282P1G3-related nucleic acid. In view of the expression of 282P1G3, cancer vaccines prevent and/or treat 282P1G3-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 282P1G3-related protein, or a 282P1G3-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 282P1G3 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 282P1G3 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 282P1G3 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 282P1G3 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 282P1G3 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.

In patients with 282P1G3-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within 282P1G3 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University, and BIMAS; SYFPEITHI on the World Wide Web. In a preferred embodiment, a 282P1G3 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table W (D), or Table W (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table W (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 282P1G3 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 282P1G3 in a host, by contacting the host with a sufficient amount of at least one 282P1G3 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 282P1G3 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 282P1G3-related protein or a man-made multiepitopic peptide comprising: administering 282P1G3 immunogen (e.g. a 282P1G3 protein or a peptide fragment thereof, a 282P1G3 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 282P1G3 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 282P1G3 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 282P1G3, in order to generate a response to the target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 282P1G3. Constructs comprising DNA encoding a 282P1G3-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 282P1G3 protein/immunogen. Alternatively, a vaccine comprises a 282P1G3-related protein. Expression of the 282P1G3-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 282P1G3 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 282P1G3-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Thus, gene delivery systems are used to deliver a 282P1G3-related nucleic acid molecule. In one embodiment, the full-length human 282P1G3 cDNA is employed. In another embodiment, 282P1G3 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 282P1G3 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 282P1G3 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 282P1G3 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 282P1G3 protein. Yet another embodiment involves engineering the overexpression of a 282P1G3 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 282P1G3 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

X.B.) 282P1G3 as a Target for Antibody-Based Therapy

282P1G3 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 282P1G3 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 282P1G3-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 282P1G3 are useful to treat 282P1G3-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

282P1G3 antibodies can be introduced into a patient such that the antibody binds to 282P1G3 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 282P1G3, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 282P1G3 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 282P1G3), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-282P1G3 antibody) that binds to a marker (e.g. 282P1G3) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 282P1G3, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 282P1G3 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-282P1G3 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 282P1G3 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄ kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 282P1G3 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Although 282P1G3 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 282P1G3 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 282P1G3 imaging, or other techniques that reliably indicate the presence and degree of 282P1G3 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

Anti-282P1G3 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-282P1G3 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-282P1G3 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 282P1G3. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-282P1G3 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 282P1G3 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-282P1G3 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-282P1G3 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-282P1G3 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-282P1G3 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-282P1G3 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg W of the anti-282P1G3 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 282P1G3 expression in the patient, the extent of circulating shed 282P1G3 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 282P1G3 in a given sample (e.g. the levels of circulating 282P1G3 antigen and/or 282P1G3 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-282P1G3 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 282P1G3-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-282P1G3 antibodies that mimic an epitope on a 282P1G3-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

X.C.) 282P1G3 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 282P1G3 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 282P1G3, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 282P1G3 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-13) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by ⁵¹Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.

For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 37), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 38), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 39). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAa (SEQ ID NO: 40), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the c- and a-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to c- and a-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P₃CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising Dc Pulsed with Ctl and/or Htl Peptides

An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate

CTL responses to 282P1G3. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 282P1G3.

X.D. Adoptive Immunotherapy

Antigenic 282P1G3-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 282P1G3. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 282P1G3. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 282P1G3-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 282P1G3, a vaccine comprising 282P1G3-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administration of the anti-282P1G3 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-282P1G3 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 282P1G3 expression in the patient, the extent of circulating shed 282P1G3 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body area weekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 10⁴ cells to about 10⁶ cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹ cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶ cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸ cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 282P1G3

As disclosed herein, 282P1G3 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression analysis of 282P1G3 in normal tissues, and patient specimens”).

282P1G3 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto et al., Cancer Detect Prey 2000; 24(1):1-12). Therefore, this disclosure of 282P1G3 polynucleotides and polypeptides (as well as 282P1G3 polynucleotide probes and anti-282P1G3 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 282P1G3 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 282P1G3 polynucleotides described herein can be utilized in the same way to detect 282P1G3 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 282P1G3 polypeptides described herein can be utilized to generate antibodies for use in detecting 282P1G3 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 282P1G3 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 282P1G3-expressing cells (lymph node) is found to contain 282P1G3-expressing cells such as the 282P1G3 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 282P1G3 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 282P1G3 or express 282P1G3 at a different level are found to express 282P1G3 or have an increased expression of 282P1G3 (see, e.g., the 282P1G3 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 282P1G3) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a 282P1G3 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.

The 282P1G3 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.

The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC 1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).

Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information concerning the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 282P1G3, the 282P1G3 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 282P1G3 protein and immune responses related thereto very useful. Use of the 282P1G3 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.

Immunohistochemical reagents specific to 282P1G3 are also useful to detect metastases of tumors expressing 282P1G3 when the polypeptide appears in tissues where 282P1G3 is not normally produced.

Thus, 282P1G3 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 282P1G3 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression analysis of 282P1G3 in normal tissues, and patient specimens,” where a 282P1G3 polynucleotide fragment is used as a probe to show the expression of 282P1G3 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 November-December 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 282P1G3 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 282P1G3 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 282P1G3 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 282P1G3 polypeptide shown in FIG. 3).

As shown herein, the 282P1G3 polynucleotides and polypeptides (as well as the 282P1G3 polynucleotide probes and anti-282P1G3 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 282P1G3 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 282P1G3 polynucleotides and polypeptides (as well as the 282P1G3 polynucleotide probes and anti-282P1G3 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 282P1G3 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 282P1G3 gene maps (see the Example entitled “Chromosomal Mapping of 282P1G3” below). Moreover, in addition to their use in diagnostic assays, the 282P1G3-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

Additionally, 282P1G3-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 282P1G3. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 282P1G3 antigen. Antibodies or other molecules that react with 282P1G3 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 282P1G3 Protein Function

The invention includes various methods and compositions for inhibiting the binding of 282P1G3 to its binding partner or its association with other protein(s) as well as methods for inhibiting 282P1G3 function.

XII A) Inhibition of 282P1G3 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 282P1G3 are introduced into 282P1G3 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-282P1G3 antibody is expressed intracellularly, binds to 282P1G3 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 282P1G3 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 282P1G3 intrabodies in order to achieve the desired targeting. Such 282P1G3 intrabodies are designed to bind specifically to a particular 282P1G3 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 282P1G3 protein are used to prevent 282P1G3 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 282P1G3 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII B) Inhibition of 282P1G3 with Recombinant Proteins

In another approach, recombinant molecules bind to 282P1G3 and thereby inhibit 282P1G3 function. For example, these recombinant molecules prevent or inhibit 282P1G3 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 282P1G3 specific antibody molecule. In a particular embodiment, the 282P1G3 binding domain of a 282P1G3 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 282P1G3 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 282P1G3, whereby the dimeric fusion protein specifically binds to 282P1G3 and blocks 282P1G3 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII C) Inhibition of 282P1G3 Transcription or Translation

The present invention also comprises various methods and compositions for inhibiting the transcription of the 282P1G3 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 282P1G3 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 282P1G3 gene comprises contacting the 282P1G3 gene with a 282P1G3 antisense polynucleotide. In another approach, a method of inhibiting 282P1G3 mRNA translation comprises contacting a 282P1G3 mRNA with an antisense polynucleotide. In another approach, a 282P1G3 specific ribozyme is used to cleave a 282P1G3 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 282P1G3 gene, such as 282P1G3 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 282P1G3 gene transcription factor are used to inhibit 282P1G3 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 282P1G3 by interfering with 282P1G3 transcriptional activation are also useful to treat cancers expressing 282P1G3. Similarly, factors that interfere with 282P1G3 processing are useful to treat cancers that express 282P1G3. Cancer treatment methods utilizing such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 282P1G3 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 282P1G3 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 282P1G3 antisense polynucleotides, ribozymes, factors capable of interfering with 282P1G3 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 282P1G3 to a binding partner, etc.

In vivo, the effect of a 282P1G3 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

XIII.) Identification, Characterization and Use of Modulators of 282P1G3 Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.

In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-related Assays

Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al., J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokarnik, supra.

A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.

The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.

In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.

In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.

In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators

Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (³H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.

In this assay, labeling index with ³H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (³H)-thymidine is determined by incorporated cpm.

Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators

Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.

Use of Tumor-specific Marker Levels to Identify and Characterize Modulators

Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al.).

Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with ¹²⁵I and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators

Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 10⁶ cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.

In vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.

Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.

In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.

Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.

Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.

Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.

Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.

Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I¹²⁵, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.

Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.

Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.

Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.

Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.

In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker

As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.

Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.

XIV.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of 282P1G3 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 282P1G3 and modulating the function of 282P1G3.

The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 282P1G3 Gene

To isolate genes that are over-expressed in pancreatic cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from pancreatic cancer tissues. The 282P1G3 SSH cDNA sequence was derived from pancreatic tumor minus cDNAs derived from normal pancreas. The 282P1G3 cDNA was identified as highly expressed in the pancreas cancer.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, Pa.). mRNA for some normal tissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used:

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 41) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQ ID NO: 42) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 43) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 44) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 45) 3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 46) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQ ID NO: 47) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID NO: 48) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in pancreas cancer. The SSH reaction utilized cDNA from pancreas cancer and normal tissues.

The gene 282P1G3 sequence was derived from pancreas cancer minus normal pancreas cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from normal pancreas mixed with a pool of 9 normal tissues was used as the source of the “driver” cDNA, while the cDNA from pancreas cancer was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from normal pancreas with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μA of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:

To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min, 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 μl of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 49) and 5′ agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 50) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 bp β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 282P1G3 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 282P1G3 SSH sequence and are listed below:

282P1G3.1 (SEQ ID NO: 51) 5′-TAAGGTCTCAGCTGTAAACCAAAAG-3′ 282P1G3.2 (SEQ ID NO: 52) 5′-CTGTTTTAAGATTGTTGGAACCTGT-3′

A typical RT-PCR expression analysis is shown in FIG. 14. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas.

Example 2 Isolation of Full Length 282P1G3 Encoding cDNA

The 282P1G3 SSH cDNA sequence was derived from a subtraction consisting of pancreas cancer minus a normal pancreas. The SSH cDNA sequence of 321 bp (FIG. 1) was designated 282P1G3.

282P1G3 v.2 of 3464 bp was cloned from a pool of normal tissue cDNA library, revealing an ORF of 1171 amino acids (FIG. 2 and FIG. 3). Other variants of 282P1G3 were also identified and these are listed in FIG. 2 and FIG. 3.

282P1G3 v.1, v.9, v.10, v.11, v.24 and v.25 proteins are 1224 amino acids in length and differ from each other by one amino acid as shown in FIG. 11. 282P1G3 v.12 through v.23, v.26 and v.27 are SNP variants and code for the same protein as 282P1G3 v.1. 282P1G3 v.2, v.3, v.4, v.5, v.6, v.7, and v.8 are splice variants of 282P1G3 v.1 and code for proteins of 1171, 893, 1117, 1208, 1183, 1236, and 1195 amino acids, respectively. 282P1G3 v.28 is a splice variant identified by the 282P1G3 SSH, and deletes the second exon of v.1.

282P1G3 v.1 shows 99% identity over 7650 nucleotides to cell adhesion molecule with homology to L1CAM (close homolog of L1) (CHL1), accession number NM_(—)006614. It is a neural recognition molecule that may be involved in signal transduction pathways. 282P1G3 v.2 is a novel splice variant of 282P1G3 and has not been previously described.

Example 3 Chromosomal Mapping of 282P1G3

Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Cornell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).

282P1G3 maps to chromosome 3p26.1 using 282P1G3 sequence and the NCBI BLAST tool located on the World Wide Web.

Example 4 Expression Analysis of 282P1G3 in Normal Tissues and Patient Specimens

Expression analysis by RT-PCR demonstrated that 282P1G3 is strongly expressed in pancreas cancer and ovary cancer patient specimens (FIG. 14). First strand cDNA was prepared from (A) vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool; (B) normal stomach, normal brain, normal heart, normal liver, normal skeletal muscle, normal testis, normal prostate, normal bladder, normal kidney, normal colon, normal lung, normal pancreas, and a pool of cancer specimens from pancreas cancer patients, ovary cancer patients, and cancer metastasis specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. (A) Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas. (B) Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression in pancreas cancer, ovary cancer, cancer metastasis, and normal brain compared to all other normal tissues tested.

Extensive expression of 282P1G3 in normal tissues is shown in FIG. 15. Two multiple tissue northern blots (Clontech) both with 2 μg of mRNA/lane were probed with the 282P1G3 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 9-10 kb transcript in normal but not in any other normal tissue tested.

Expression of 282P1G3 in pancreas cancer patient specimens is shown in FIG. 16. RNA was extracted from pancreas cancer cell lines (CL), normal pancreas (N), and pancreas cancer patient tumor (T). Northern blots with 10 μg of total RNA were probed with the 282P1G3 SSH fragment. Size standards in kilobases are on the side. Results show expression of 282P1G3 in pancreas cancer patient tumor specimen but not in the cell lines nor in the normal pancreas.

Expression of 282P1G3 was also detected in ovary cancer patient specimens (FIG. 17). RNA was extracted from ovary cancer cell lines (CL), normal ovary (N), and ovary cancer patient tumor (T). Northern blots with 10 μg of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in ovary cancer patient tumor specimen but not in the cell lines nor in the normal ovary.

FIG. 18 shows expression of 282P1G3 in lymphoma cancer patient specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10 μg of total RNA were probed with the 282P1G3 sequence. Size standards in kilobases are on the side. Results show expression of 282P1G3 in lymphoma patient specimens but not in the normal blood cells tested.

The restricted expression of 282P1G3 in normal tissues and the expression detected in cancer patient specimens suggest that 282P1G3 is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 282P1G3

Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail and GenScan on the World Wide Web. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 282P1G03 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 282P1G03 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.

Using the full-length gene and EST sequences, eight additional transcript variants were identified, designated as 282P1G03 v.2, v.3, v.4, v.5, v.6, v.7, v.8 and v.28. The boundaries of exons in the original transcript, 282P1G03 v.1 were shown in Table LI. FIG. 12 shows the structures of the transcript variants. Theoretically, each different combination of exons in spatial order (aligned on the genomic sequence), e.g. exons 2, 3, 5, 7, and 9-28 of v.1, is a potential splice variant.

Tables LII(a)-(h) through LV(a)-(h) are set forth on a variant-by-variant bases. Tables LII(a)-(h) show the nucleotide sequence of the transcript variant. Tables LIII(a)-(h) show the alignment of the transcript variant with nucleic acid sequence of 282P1G03 v.1. Tables LIV(a)-(h) show the amino acid translation of the transcript variant for the identified reading frame orientation. Tables LV(a)-(h) display alignments of the amino acid sequence encoded by the splice variant with that of 282P1G03 v.1.

Example 6 Single Nucleotide Polymorphisms of 282P1G3

A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNP are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).

Using the methods described above, 19 SNP were identified in the original transcript, 282P1G03 v.1, at positions 320 (c/t), 668 (c/t), 1178 (a/g), 3484 (c/t), 4615 (g/a), 4636 (-/t), 5078 (c/t), 5530 (t/a), 5812 (c/t), 6114 (a/g), 6229 (c/t), 6383 (g/a), 6626 (c/t), 6942 (c/t), 7085 (c/t), 2684 (a/g), 3864 (t/c), 5768 (t/c) and 6125 (c/t). The transcripts or proteins with alternative allele were designated as variant 282P1G03 v.9 through v.25, as shown in FIG. 10. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in FIG. 11. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 282P1G03 v.2) that contains the site of the SNP.

Example 7 Production of Recombinant 282P1G3 in Prokaryotic Systems

To express recombinant 282P1G3 and 282P1G3 variants in prokaryotic cells, the full or partial length 282P1G3 and 282P1G3 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 282P1G3 variants are expressed: the full length sequence presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 282P1G3, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII:

To generate 282P1G3 sense and anti-sense RNA probes for RNA in situ investigations, pCR11 constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 282P1G3 cDNA. The pCR11 vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 282P1G3 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 282P1G3 at the RNA level. Transcribed 282P1G3 RNA representing the cDNA amino acid coding region of the 282P1G3 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 282P1G3 protein.

B. Bacterial Constructs:

pGEX Constructs:

To generate recombinant 282P1G3 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 282P1G3 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus. The GST and 6×His Tag5 permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6×His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 282P1G3-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs:

To generate, in bacteria, recombinant 282P1G3 proteins that are fused to maltose-binding protein (MBP), all or parts of the 282P1G3 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 282P1G3 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×His Tag5 permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6×His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 282P1G3. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs:

To express 282P1G3 in bacterial cells, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 282P1G3 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 282P1G3 protein are expressed as amino-terminal fusions to NusA.

C. Yeast Constructs:

pESC Constructs:

To express 282P1G3 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope Tag5 in the same yeast cell. This system is useful to confirm protein-protein interactions of 282P1G3. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations that are found when expressed in eukaryotic cells.

pESP Constructs:

To express 282P1G3 in the yeast species Saccharomyces pombe, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 282P1G3 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 282P1G3 in Higher Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 282P1G3 in eukaryotic cells, the full or partial length 282P1G3 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 282P1G3 were expressed in these constructs, amino acids 1 to 1224, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 282P1G3 v.1, and v.9 through v.25; amino acids 1 to 1171, 1 to 893, 1 to 1117, 1 to 1208, 1 to 1183, 1 to 1236, 1 to 1195 of v.2, v.3, v.4, v.5, v.6, v.7, and v.8 respectively; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 282P1G3 variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-282P1G3 polyclonal serum, described herein.

pcDNA4/HisMax Constructs:

To express 282P1G3 in mammalian cells, a 282P1G3 ORF, or portions thereof, of 282P1G3 are cloned into pcDNA4/H isMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6×His) epitopes fused to the amino-terminus. The pcDNA4/H isMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs:

To express 282P1G3 in mammalian cells, a 282P1G3 ORF, or portions thereof, of 282P1G3 with a consensus Kozak translation initiation site is cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

The complete ORF of 282P1G3 v.2 was cloned into the pcDNA3.1/MycHis construct to generate 282P1G3.pcDNA3.1/MycHis. FIG. 19 shows expression of 282P1G3.pcDNA3.1/MycHis following transfection into 293T cells. 293T cells were transfected with either 282P1G3.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control. Forty hours later, cell lysates were collected. Samples were run on an SD S-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression of 282P1G3 from the 282P1G3.pcDNA3.1/MycHis construct in the lysates of transfected cells.

pcDNA3.1/CT-GFP-TOPO Construct:

To express 282P1G3 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 282P1G3 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 282P1G3 protein.

PAPtag:

A 282P1G3 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 282P1G3 protein while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of a 282P1G3 protein. The resulting recombinant 282P1G3 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 282P1G3 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pTag5:

A 282P1G3 ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 282P1G3 protein with an amino-terminal IgGκ signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 282P1G3 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 282P1G3 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

The extracellular domain, amino acids 26-1043, of 282P1G3 v.2 was cloned into the pTag5 construct to generate 282P1G3.pTag5. FIG. 20 shows expression and secretion of the extracellular domain of 282P1G3 following 282P1G3.pTag5 vector transfection into 293T cells. 293T cells were transfected with 282P1G3.pTag5 construct. Forty hours later, supernatant as well as cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression and secretion of 282P1G3 from the 282P1G3.pTag5 transfected cells.

PsecFc:

A 282P1G3 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 282P1G3 proteins, while fusing the IgGK signal sequence to N-terminus. 282P1G3 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 282P1G3 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 282P1G3 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRα Constructs:

To generate mammalian cell lines that express 282P1G3 constitutively, 282P1G3 ORF, or portions thereof, of 282P1G3 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 282P1G3, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 282P1G3 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 53) is added to cloning primer at the 3′ end of the ORF. Additional pSRa constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 282P1G3 proteins.

Additional Viral Vectors:

Additional constructs are made for viral-mediated delivery and expression of 282P1G3. High virus titer leading to high level expression of 282P1G3 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 282P1G3 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 282P1G3 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems:

To control expression of 282P1G3 in mammalian cells, coding sequences of 282P1G3, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 282P1G3. These vectors are thereafter used to control expression of 282P1G3 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 282P1G3 proteins in a baculovirus expression system, 282P1G3 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-282P1G3 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 282P1G3 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 282P1G3 protein can be detected using anti-282P1G3 or anti-His-tag antibody. 282P1G3 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 282P1G3.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), and FIG. 9(A-C) depict graphically five amino acid profiles of 282P1G3 variants 1, 3, and 7, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.p1) on the ExPasy molecular biology server.

These profiles: FIG. 5(A-C), Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6(A-C), Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7(A-C), Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8(A-C), Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9(A-C), Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 282P1G3 variant proteins. Each of the above amino acid profiles of 282P1G3 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.

Antigenic sequences of the 282P1G3 variant proteins indicated, e.g., by the profiles set forth in FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), and/or FIG. 9(A-C) are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-282P1G3 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 282P1G3 protein variants listed in FIGS. 2 and 3, In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 282P1G3 protein variants 1 through 8, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (Guermeur, 1997, accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/)). The analysis indicates that 282P1G3 variant 1 is composed of 15.77% alpha helix, 26.14% extended strand, and 58.09% random coil (FIG. 13A). Variant 2 is composed of 14.86% alpha helix, 26.39% extended strand, and 58.75% random coil (FIG. 13B). Variant 3 is composed of 14.00% alpha helix, 29.34% extended strand, and 56.66% random coil (FIG. 13C). Variant 4 is composed of 15.94% alpha helix, 26.14% extended strand, and 57.92% random coil (FIG. 13D). Variant 5 is composed of 15.73% alpha helix, 26.32% extended strand, and 57.95% random coil (FIG. 13E). Variant 6 is composed of 16.99% alpha helix, 25.36% extended strand, and 57.65% random coil (FIG. 13F). Variant 7 is composed of 15.78% alpha helix, 26.13% extended strand, and 58.09% random coil (FIG. 13G). Variant 8 is composed of 16.99% alpha helix, 25.36% extended strand, and 57.66% random coil (FIG. 13H).

Analysis for the potential presence of transmembrane domains in the 282P1G3 variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Shown graphically in FIGS. 13I and 13J are the results of analysis of variant 1 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13I) and 1 transmembrane domain using the TMHMM program (FIG. 13J). Shown graphically in FIGS. 13K and 13L are the results of analysis of variant 2 depicting the presence and location of 1 transmembrane domains using the TMpred program (FIG. 13K) and 1 transmembrane domain using the TMHMM program (FIG. 13L). Shown graphically in FIGS. 13M and 13N are the results of analysis of variant 3 depicting no transmembrane domain using both the TMpred program (FIG. 13M) and TMHMM program (FIG. 13N). Shown graphically in FIGS. 13O and 13P are the results of analysis of variant 4 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13O) and 1 transmembrane domain using the TMHMM program (FIG. 13P). Shown graphically in FIGS. 13Q and 13R are the results of analysis of variant 5 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13Q) and 1 transmembrane domain using the TMHMM program (FIG. 13R). Shown graphically in FIGS. 13S and 13T are the results of analysis of variant 6 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13S) and 1 transmembrane domain using the TMHMM program (FIG. 13T). Shown graphically in FIGS. 13U and 13V are the results of analysis of variant 7 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13U) and 1 transmembrane domain using the TMHMM program (FIG. 13V). Shown graphically in FIGS. 13W and 13X are the results of analysis of variant 8 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13W) and 1 transmembrane domain using the TMHMM program (FIG. 13X). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table VI.

Example 10 Generation of 282P1G3 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 282P1G3 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5(A-C), FIG. 6(A & C), FIG. 7(A-C), FIG. 8(A-C), or FIG. 9(A-C) for amino acid profiles that indicate such regions of 282P1G3 protein variants).

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 282P1G3 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 11. For example, in 282P1G3 variant 1, such regions include, but are not limited to, amino acids 57-75, amino acids 131-135, amino acids 210-265, amino acids 550-588, and amino acids 662-688. In sequence unique to variant 3, such regions include, but are not limited to, amino acids 855-872 and amino acids 856-886. In sequence specific for variant 7, such regions include, but are not limited to, amino acids 345-356. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 57-75 of 282P1G3 variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 282P1G3 variant proteins, analogs or fusion proteins thereof. For example, the 282P1G3 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. In another embodiment, amino acids 26-265 of 282P1G3 variant 1 was fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 282P1G3 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems”), and retains post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 26-1,043 of variant 2, encoding the extracellular domain, was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 282P1G3 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with the Tag5-282P1G3 variant 2 protein, the full-length 282P1G3 variant 1 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-282P1G3 serum and with anti-His antibody (See FIG. 19; Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 282P1G3 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 282P1G3-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 282P1G3 are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 282P1G3 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-282P1G3 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-282P1G3 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11 Generation of 282P1G3 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 282P1G3 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 282P1G3 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 282P1G3 protein variant sequence, regions of the 282P1G3 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), or FIG. 9(A-C), and the Example entitled “Antigenicity Profiles and Secondary Structure”) Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 282P1G3 variant, such as 293T-282P1G3 variant 1 or 300.19-282P1G3 variant 1 murine Pre-B cells, are used to immunize mice.

To generate mAbs to a 282P1G3 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ 282P1G3-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 282P1G3 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 26-1,043 of variant 2 was cloned into the Tag5 mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 282P1G3 variant 2 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 282P1G3 variant.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 282P1G3 monoclonal antibodies, a Tag5-282P1G3 variant 2 antigen encoding amino acids 26-1,043, was expressed (FIG. 20) and then purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5-282P1G3 variant 2 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 282P1G3 variant 2 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 282P1G3 variant 2 cDNA (see e.g., the Example entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems” and FIG. 19). Other recombinant 282P1G3 variant 2-expressing cells or cells endogenously expressing 282P1G3 variant 2 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 282P1G3 specific antibody-producing clones.

To generate monoclonal antibodies that are specific for each 282P1G3 variant protein, immunogens are designed to encode sequences unique for each variant. For example, peptides or recombinant protein antigens (i.e. Tag5 fusion proteins) encompassing the unique sequence derived from alternate exon usage in splice variants 2, 3, 4, 5, 6, and 7 are used as immunogens. In one embodiment, a Tag5 protein encoding amino acids 838-893 unique to 282P1G3 variant 3 is produced, purified, and used as immunogen to derive monoclonal antibodies specific to 282P1G3 variant 3. In another embodiment, an antigenic peptide composed of amino acids 1025-1037 of 282P1G3 variant 2 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 817-829 of 282P1G3 variant 4 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 220-232 of 282P1G3 variant 5 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 122-134 of 282P1G3 variant 6 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 339-362 of 282P1G3 variant 7 is coupled to KLH and used as immunogen. Hybridoma supernatants are then screened on the respective antigen and then further screened on cells expressing the specific variant and cross-screened on cells expressing the other variants to derive variant-specific monoclonal antibodies.

The binding affinity of a 282P1G3 variant monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 282P1G3 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measured IC₅₀ values are reasonable approximations of the true K_(D) values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC₅₀ of a positive control for inhibition by the IC₅₀ for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC₅₀ nM values by dividing the IC₅₀ nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 282P1G3 set forth in FIGS. 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 282P1G3 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount j_(i) to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of j_(i). For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 282P1G3 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 282P1G3 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦500 nM, often ≦200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 282P1G3 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC₅₀ of ≦500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 282P1G3 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC):

PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide:

CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the Detacha-bead® reagent. Typically about 200-250×10⁶ PBMC are processed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×10⁶ cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml Detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting Up Induction Cultures:

0.25 ml cytokine-generated DC (at 1×10⁵ cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶ cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.

Restimulation of the Induction Cultures with Peptide Pulsed Adherent Cells:

Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a ⁵¹Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) ⁵¹Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 10⁶ per ml and diluted 1:10 with K₅₆₂ cells at a concentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr release sample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous ⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates are washed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H₃PO₄ and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flask containing the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×10⁶/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assay or at 1×10⁶/ml in the in situ IFNγ assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25 flask containing the following: 1×10⁶ autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 282P1G3. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology.

Example 15 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of 5000 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate 500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 282P1G3-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.

Example 16 Identification and Confirmation of 282P1G3-Derived Sequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 282P1G3-derived, HLA class II HTL epitopes, a 282P1G3 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (L e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 282P1G3-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 282P1G3-derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 282P1G3 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 17 Immunogenicity of 282P1G3-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 282P1G3-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table W (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.

Example 19 CTL Recognition of Endogenously Processed Antigens after Priming

This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells in the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 282P1G3 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 282P1G3 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/K^(b) transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 282P1G3-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 282P1G3-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

Immunization Procedures:

Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/K^(b) mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell Lines:

Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)

In Vitro CTL Activation:

One week after priming, spleen cells (30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

Assay for Cytotoxic Activity:

Target cells (1.0 to 1.5×10⁶) are incubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 10⁴ ⁵¹Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100× (experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specific lytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptide is subtracted from the lytic units/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Cr release is obtained at the effector (E):target (T) ratio of 50:1 (i.e., 5×10⁵ effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a 282P1G3-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responses that are correlated with 282P1G3 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 282P1G3. For example, if it has been observed that patients who spontaneously clear 282P1G3-expressing cells generate an immune response to at least three (3) epitopes from 282P1G3 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site on the World Wide Web.

In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 282P1G3, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 282P1G3.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 282P1G3, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 282P1G3 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and H is antibody epitope tag coded for by the pcDNA 3.1 Myc-H is vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μA reactions containing Pfu polymerase buffer (1x=10 mM KCL, 10 mM (NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it Induces Immunogenicity

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a ⁵¹Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-A^(b)-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent 282P1G3 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 282P1G3-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 282P1G3-associated disease.

Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 282P1G3 Sequences

A native 282P1G3 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 282P1G3 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 282P1G3, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.

Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 282P1G3 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 282P1G3 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 282P1G3 as well as tumor-associated antigens that are often expressed with a target cancer associated with 282P1G3 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 282P1G3. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 282P1G3 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 282P1G3 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain>99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 282P1G3 epitope, and thus the status of exposure to 282P1G3, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 282P1G3-associated disease or who have been vaccinated with a 282P1G3 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 282P1G3 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Viral. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release-spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 282P1G3 or a 282P1G3 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 282P1G3 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for ³H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of ³H-thymidine incorporation in the presence of antigen divided by the ³H-thymidine incorporation in the absence of antigen.

Example 29 Induction Of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials In Patients Expressing 282P1G3

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 282P1G3. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 282P1G3, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:

The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 282P1G3.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 282P1G3-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 282P1G3 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells (DC)

Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 282P1G3 protein from which the epitopes in the vaccine are derived.

For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2−50×10⁶ DC per patient are typically administered, larger number of DC, such as 10⁷ or 10⁸ can also be provided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 10⁸ to 10¹⁰. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×10⁶ DC, then the patient will be injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 282P1G3 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 282P1G3. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 282P1G3 to isolate peptides corresponding to 282P1G3 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 282P1G3-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 282P1G3. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 282P1G3. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 282P1G3-encoding transcript.

Example 35 Purification of Naturally-Occurring or Recombinant 282P1G3 Using 282P1G3-Specific Antibodies

Naturally occurring or recombinant 282P1G3 is substantially purified by immunoaffinity chromatography using antibodies specific for 282P1G3. An immunoaffinity column is constructed by covalently coupling anti-282P1G3 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 282P1G3 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 282P1G3 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/282P1G3 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 36 Identification of Molecules Which Interact with 282P1G3

282P1G3, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 282P1G3, washed, and any wells with labeled 282P1G3 complex are assayed. Data obtained using different concentrations of 282P1G3 are used to calculate values for the number, affinity, and association of 282P1G3 with the candidate molecules.

Example 37 In Vivo Assay for 282P1G3 Tumor Growth Promotion

The effect of the 282P1G3 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 282P1G3. For example, SCID mice are injected subcutaneously on each flank with 1×10⁶ of either 3T3, ovarian (e.g. PA-1 cells), pancreatic (e.g. Panc-1 cells) or lymphoma (e.g. Daudi cells) cancer cell lines containing tkNeo empty vector or 282P1G3. At least two strategies may be used: (1) Constitutive 282P1G3 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and followed over time to determine if 282P1G3-expressing cells grow at a faster rate and whether tumors produced by 282P1G3-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cells orthotopically to determine if 282P1G3 has an effect on local growth in the pancreas, and whether 282P1G3 affects the ability of the cells to metastasize, specifically to lymph nodes, and bone (Mild, T et al., Oncol Res. 2001; 12:209; Fu X et al., Int J. Cancer. 1991, 49:938). The effect of 282P1G3 on bone tumor formation and growth may be assessed by injecting tumor cells intratibially.

The assay is also useful to determine the 282P1G3 inhibitory effect of candidate therapeutic compositions, such as, 282P1G3 intrabodies, 282P1G3 antisense molecules and ribozymes.

Example 38 282P1G3 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo

The significant expression of 282P1G3 in cancer tissues and surface localization, together with its restrictive expression in normal tissues makes 282P1G3 a good target for antibody therapy. Similarly, 282P1G3 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-282P1G3 mAbs in human cancer xenograft mouse models, including ovarian, pancreatic or lymphoma and other -282P1G3 cancers listed in Table I, is evaluated by using recombinant cell lines such as Pa-1-282P1G3, Panc1-282P1G3, Daudi-282P1G3, and 3T3-282P1G3 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): 16-23), as well as human xenograft models (Saffran et al. PNAS 1999, 10:1073-1078).

Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic ovary, pancreas, or blood cancer xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-282P1G3 mAbs inhibit formation of tumors in mouse xenografts. Anti-282P1G3 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-282P1G3 mAbs in the treatment of local and advanced stages several solid tumors. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or on the World Wide Web.

Administration of the anti-282P1G3 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 282P1G3 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-282P1G3 mAbs for the treatment of local and metastatic cancer. This example indicates that unconjugated 282P1G3 monoclonal antibodies are effective to inhibit the growth of human pancreatic, ovarian and lymphomas tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 282P1G3 mAbs

Materials and Methods:

282P1G3 Monoclonal Antibodies:

Monoclonal antibodies are raised against 282P1G3 as described in the Example entitled “Generation of 282P1G3 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 282P1G3. Epitope mapping data for the anti-282P1G3 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 282P1G3 protein. Immunohistochemical analysis of cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of PC3, UM-UC3, CaKl and A427 tumor xenografts.

Cell Lines and Xenografts

The cancer cell lines PA-1, Panc1, Daudi cell lines, as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in DMEM supplemented with L-glutamine and 10% FBS.

PA1-282P1G3, Panc1-282P1G3, Daudi-282P1G3 and 3T3-282P1G3 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): 14523.

Human patient-derived xenografts are passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., Nat. Med. 1999, 5:280). Single-cell suspensions of tumor cells are prepared as described in Craft, et al.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 2×10⁶ cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.e. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as length×width×height. Mice with Subcutaneous tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. Following tumor implantation, the mice are segregated into groups for the appropriate treatments, with anti-282P1G3 or control mAbs being injected i.p. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure hCG levels.

Anti-282P1G3 mAbs Inhibit Growth of 282P1G3-Expressing Xenograft-Cancer Tumors

The effect of anti-282P1G3 mAbs on tumor formation is tested by using cell line (e.g. PA-1, Panel, Daudi and 3T3) and patient-derived tumor orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse organ results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-CK20 for prostate cancer (Lin S et al., Cancer Detect Prey. 2001; 25:202).

Another advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth is partly dependent on new blood vessel development. Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovasculature is regulated by the xenograft tumor (Davidoff A M et al., Clin Cancer Res. 2001; 7:2870; Solesvik O et al., Eur J Cancer Clin Oncol. 1984, 20:1295). The effect of antibody and small molecule on neovascularization is studied in accordance with procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment.

Mice bearing established orthotopic tumors are administered 1000 μg injections of either anti-282P1G3 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-282P1G3 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-282P1G3 antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-282P1G3 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-282P1G3 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-282P1G3 Antibodies in Humans

Anti-282P1G3 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-282P1G3 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 282P1G3 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-282P1G3 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-282P1G3 mAb specifically binds to carcinoma cells. Thus, anti-282P1G3 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 282P1G3. Shedding or release of an extracellular domain of 282P1G3 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 282P1G3 by anti-282P1G3 antibodies in serum and/or urine samples from suspect patients.

Anti-282P1G3 antibodies that specifically bind 282P1G3 are used in therapeutic applications for the treatment of cancers that express 282P1G3. Anti-282P1G3 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-282P1G3 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “282P1G3 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-282P1G3 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-282P1G3 Antibodies In Vivo

Antibodies are used in accordance with the present invention which recognize an epitope on 282P1G3, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 282P1G3 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-282P1G3 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-282P1G3 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-282P1G3 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

II.) Monotherapy: In connection with the use of the anti-282P1G3 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I¹³¹, Y⁹⁰) to anti-282P1G3 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 282P1G3. In connection with the use of the anti-282P1G3 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (¹¹¹In)-282P1G3 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 282P1G3 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-282P1G3 antibodies can be administered with doses in the range of 5 to 400 mg/m², with the lower doses used, e.g., in connection with safety studies. The affinity of anti-282P1G3 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-282P1G3 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-282P1G3 antibodies can be lower, perhaps in the range of 50 to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-282P1G3 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-282P1G3 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-282P1G3 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 282P1G3 expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 282P1G3. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-282P1G3 antibodies are found to be safe upon human administration.

Example 41 Human Clinical Trial Adjunctive Therapy with Human Anti-282P1G3 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-282P1G3 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-282P1G3 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-282P1G3 antibody with dosage of antibody escalating from approximately about 25 mg/m² to about 275 mg/m² over the course of the treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275 mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, L e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 282P1G3. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-282P1G3 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-282P1G3 Antibody

Anti-282P1G3 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-282P1G3 antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-282P1G3 Antibody

Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-282P1G3 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44 Comparison of 282P1G3 to Known Sequences

The human 282P1G3 protein exhibit a high degree of homology to a known human protein, cell adhesion molecule with homology to L1CAM precursor (gi 27894376), also known Close Homolog of L1 (CHL1) or CALL. Human CHL1 shows 99% identity to 282P1G3 at the protein level (FIG. 4A). The mouse homolog of 282P1G3 has been identified as murine CHL1 (gi 6680936), and shows 82% identity and 89% homology to 282P1G3 (FIG. 4B). CHL1 has been reported to regulate neuronal development by altering cell adhesion and axonal projections (Montag-Sallaz M et al., Mol. Cell. Biol. 2002, 22:7967). In addition, CHL1 was found to play a role in neurite growth and survival (Dong L et al., J. Neurosci. Res, 2002; Chaisuksunt V et al., J. Comp. Neurol 2000, 425:382). Mutations in CHL1 have been associated with schizophrenia and metal disorders (Sakurai et al., Mol Psychiatry 2002, 7:412; We H et al., Hum Genet. 1998, 103:355).

The prototype member of the 282P1G3 family, 282P1G3v.1, is a 1224 amino acids protein. Initial bioinformatics analysis using topology prediction programs suggested that 191 P2D 14 may contain 2 transmembranes based on hydrophobicity profile. However, the first hydrophobic domain was identified as a signal sequence, rendering 191P2D12 a single transmembrane protein.

The 282P1G3 gene has several variants, including 5 SNP represented by 282P1G3 v.9, v.10, v.11, v.24 and v.25. In addition, several splice variants have been identified, including deletion variants such as 282P1G3 v.2, v.4, v.5 and v.6, as well as insertion mutants such as 282P1G3 v.7 and v.8, and a splice variant at aa 838 of 282P1G3 v.1, namely 282P1G3 v.3.

Motif analysis revealed the presence of several protein functional motifs in the 282P1G3 protein (Table L). Six immunoglobulin domains have been identified in addition to four fibronectin type III repeats Immunoglobulin domains are found in numerous proteins and participate in protein-protein such including protein-ligand interactions (Weismann et al., J Mol Med 2000, 78:247). In addition, Ig-domains function in cell adhesion, allowing the interaction of leukocytes and blood-born cells with the endothelium (Wang and Springer, Immunol Rev 1998, 163:197). Fibronectin type III repeats are 100 amino acid domains with binding sites for various molecules, including DNA, heparin, basement membrane and cell surface proteins (Kimizuka et al., J Biol. Chem. 1991, 266:3045; Yokosaki et al., J Biol. Chem. 1998, 273:11423). The majority for proteins containing fibronectin III motifs participate in cell surface binding, binding to specific substrates including heparin, collagen, DNA, actin and fibrin, or are involved in binding to fibronectin receptors. Fibronectins have been reported to function in wound healing; cell adhesion, cell differentiation, cell migration and tumour metastasis (Bloom et al., Mol Biol Cell. 1999, 10:1521; Brodt P. Cancer Met Rev 1991, 10:23). The motifs found in 282P1G3 as well as its similarity to CHL1 indicate that 282P1G3 can participate in tumor growth and progression by enhancing the initial stages of tumorigenesis, including tumor establishment and tumor growth, by allowing adhesion to basement membranes and surrounding cells, by mediating cell migration and metastasis.

Accordingly, when 282P1G3 functions as a regulator of tumor establishment, tumor formation, tumor growth, survival or cell signaling, 282P1G3 is used for therapeutic, diagnostic, prognostic and/or preventative purposes. In addition, when a molecule, such as a splice variant or SNP of 282P1G3 is expressed in cancerous tissues, such as those listed in Table I, they are used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Example 45 Regulation of Transcription

The cell surface localization of 282P1G3 coupled to the presence of Ig-domains within its sequence indicate that 282P1G3 modulates signal transduction and the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 282P1G3. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 282P1G3-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J. Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS, androgen or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.

Thus, 282P1G3 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46 Identification and Confirmation of Potential Signal Transduction Pathways

Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J. Neurochem. 2001; 76:217-223). Immunoglobulin-like molecules in particular has been associated with several tyrpsine kinases including Lyc, Blk, syk (Tamir and Cambier, Oncogene. 1998, 17:1353), the MAPK signaling cascade that control cell mitogenesis and calcium flux (Vilen J et al., J Immunol 1997, 159:231; Jiang F, Jia Y, Cohen I. Blood. 2002, 99:3579). In addition, the 282P1G3 protein contains several phosphorylation sites (see Table VI) indicating an association with specific signaling cascades. Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 282P1G3 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 282P1G3, including phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, catenin, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).). In order to determine whether expression of 282P1G3 is sufficient to regulate specific signaling pathways not otherwise active in resting cancer cells, the effect of 282P1G3 on the activation of the signaling cascade is investigated in the cancer cell lines PA-1, Panel and Daudi. Cancer cells stably expressing 282P1G3 or neo are stimulated with growth factor, FBS or other activating molecules. Whole cell lysates are analyzed by western blotting.

To confirm that 282P1G3 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion

Gene-mediated effects can be assayed in cells showing mRNA expression.

Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.

Signaling pathways activated by 282P1G3 are mapped and used for the identification and validation of therapeutic targets. When 282P1G3 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

Based on the role of Ig-domains and fibronectin motifs in cell growth and signal transduction, the 282P1G3 gene can contribute to the growth, invasion and transformation of cancer cells. The role of 282P1G3 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate cell lines, as well as NIH 3T3 cells engineered to stably express 282P1G3. Parental cells lacking 282P1G3 and cells expressing 282P1G3 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).

To confirm the role of 282P1G3 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 282P1G3 are compared to N1H-3T3 cells expressing 282P1G3, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60:6730).

To confirm the role of 282P1G3 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including prostate, breast and kidney cell lines lacking 282P1G3 are compared to cells expressing 282P1G3. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.

282P1G3 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 282P1G3 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 282P1G3, including normal and tumor prostate cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, taxol, etc, and protein synthesis inhibitors, such as cycloheximide Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 282P1G3 can play a critical role in regulating tumor progression and tumor load.

When 282P1G3 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of fibronectins on tumor cell adhesion and their interaction with endothelial cells, 282P1G3 plays a role in angiogenesis (Mareel and Leroy: Physiol Rev, 83:337; DeFouw L et al., Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 282P1G3 in angiogenesis, enhancement or inhibition, is confirmed.

For example, endothelial cells engineered to express 282P1G3 are evaluated using tube formation and proliferation assays. The effect of 282P1G3 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 282P1G3 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 282P1G3 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 49 Involvement in Protein-Protein Interactions

Ig-domains and fibronectin motifs have been shown to mediate interaction with other proteins, including cell surface protein. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 282P1GJ. Immunoprecipitates from cells expressing 282P1G3 and cells lacking 282P1G3 are compared for specific protein-protein associations.

Studies are performed to confirm the extent of association of 282P1G3 with effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates etc. Studies comparing 282P1G3 positive and 282P1G3 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr. Opin. Chem. Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 282P1G3-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 282P1G3, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 282P1G3.

Thus, it is found that 282P1G3 associates with proteins and small molecules. Accordingly, 282P1G3 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 50 Involvement of 282P1G3 in Cell-Cell Communication

Cell-cell communication is essential in maintaining organ integrity and homeostasis, both of which become deregulated during tumor formation and progression. Based on the presence of a fibronectin motif in 282P1G3, a motif known to be involved in cell interaction and cell-cell adhesion, 282P1G3 can regulate cell communication. Intercellular communications can be measured using two types of assays (J. Biol. Chem. 2000, 275:25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing 282P1G3 are compared to controls that do not express 282P1G3, and it is found that 282P1G3 enhances cell communications. Small molecules and/or antibodies that modulate cell-cell communication mediated by 282P1G3 are used as therapeutics for cancers that express 282P1G3. When 282P1G3 functions in cell-cell communication and small molecule transport, it is used as a target or marker for diagnostic, prognostic, preventative and/or therapeutic purposes.

Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Tables:

TABLE I Tissues that Express 282P1G3: a. Malignant Tissues Pancreas Ovary Lymph node

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION C Terminus 2 (Primary 3 (Primary (Primary Anchor) Anchor) Anchor) SUPERMOTIF APP TI LVMS FWY Anesthetic LIVM ATQ IV MATL Anesthesia VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS APP TSM Y APP DE AS Y A2.1 LM VQIAT V LIMAT Anesthesia LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YFWM FLIW A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs 1°anchor 1° anchor MOTIFS 1 2 3 4 5 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1° anchor 2 3 1° anchor 5 1° anchor 1 4 6 Motif a LIVMFY D preferred Motif b LIVMFAY DNQEST KRH preferred DR MFLIV VMSTA Supermotif WY CPLI Italicized residues indicate less preferred or “tolerated” residues

TABLE IV (D) HLA Class I Supermotifs SUPER- C- MOTIFS POSITION: 1 2 3 4 5 6 7 8 terminus APP 1° 1° Anchor Anchor TILVMS FWY Anesthetic 1° 1° Anchor Anchor LIVMATQ LIVMAT Anesthesia Preferred 1° YFW YFW YFW P 1° Anchor (4/5) (3/5) (4/5) (4/5) Anchor VSMATLI RK deleterious DE DE (3/5); (4/5) P (5/5) A24 1° 1° Anchor Anchor YFWIVLMT FIYWLM B7 Preferred FWY 1° FWY FWY 1° Anchor (5/5) Anchor (4/5) (3/5) VILFMWYA LIVM P (3/5) deleterious DE DE G QN DE (3/5); (3/5) (4/5) (4/5) (4/5) P(5/5); G(4/5); A(3/5); QN(3/5) B27 1° 1° Anchor Anchor FYLWMIVA RHK B44 1° 1° Anchor Anchor ED FWYLIMVA B58 1° 1° Anchor Anchor ATS FWYLIVMA B62 1° 1° Anchor Anchor QLIVMP FWYMIVLA Italicized residues indicate less preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs 9 or C- C- POSITION 1 2 3 4 5 6 7 8 terminus terminus A1 prefer- GFYW l° Anchor DEA YFW P DEQN YFW l° Anchor 9-mer red STM Y delete- DE RHKLIVAMP G A rious A1 prefer- GRHK ASTCLIVM l° Anchor GSTC ASTC LIVM DE 9-mer red DEAS Y delete- A RHKDEPY DE PQN RHK PG GP rious FW A1 prefer- YFW l° Anchor DEAQN A YFWQN PAST CGDE P l° Anchor 10-mer red STM Y delete- GP RHKGLIVM DE RHK QNA RHKYFW RHK A rious A1 prefer- YFW STCLIVM l° Anchor A YFW PG G YFW l° Anchor 10-mer red DEAS Y delete- RHK RHKDEPYFW P G PRHK QN rious A2.1 prefer- YFW l° Anchor YFW STC YFW A P l° Anchor 9-mer red LMIVQAT VLIMAT delete- DEP DERKH RKH DERKH rious C- POSITION: 1 2 3 4 5 6 7 8 9 Terminus A2.1 prefer- AYFW l° Anchor LVIM G G FYWLVIM l° Anchor 10-mer red LMIVQAT VLIMAT delete- DEP DE RKHA P RKH DERKH RKH rious A3 prefer- RHK l° Anchor YFW PRHKYFW A YFW P l° Anchor red LMVISATFC KYRHFA GD delete- DEP DE rious A11 prefer- A l° Anchor YFW YFW A YFW YFW P l° Anchor red VTLMISAGN KRYH CDF delete- DEP A G rious A24 prefer- YFWRHK l° Anchor STC YFW YFW l° Anchor 9-mer red YFWM FLIW delete- DEG DE G QNP DERHK G AQN rious A24 Prefer- l° Anchor P YFWP P l° Anchor 10-mer red YFWM FLIW Delete- GDE QN RHK DE A QN DEA rious 9 or C- C- POSITION 1 2 3 4 5 6 7 8 terminus terminus A31  Prefer- RHK l° Anchor YFW P YFW YFW AP l° Anchor 01 red MVTALIS RK Delete- DEP DE ADE DE DE DE rious A33  Prefer- l° Anchor YFW AYFW l° Anchor 01 red MVALFIST RK Delete- GP DE rious A68 Prefer- YFWSTC l° Anchor YFWLIVM YFW P l° Anchor 01 red AVTMSLI RK delete- GP DEG RHK A rious B07 Prefer- RHKFWY l° Anchor RHK RHK RHK RHK PA l° Anchor 02 red P LMFWYAIV delete- DEQNP DEP DE DE GDE QN DE rious B35 Prefer- FWYLIVM l° Anchor FWY FWY l° Anchor 01 red P LMFWYIVA delete- AGP G G rious B51 Prefer- LIVMFWY l° Anchor FWY STC FWY G FWY l° Anchor red P LIVFWYAM delete- AGPDERH DE G DEQN GDE rious KSTC B53 prefer- LIVMFWY l° Anchor FWY STC FWY LIVMFWY FWY l° Anchor 01 red P IMFWYALV delete- AGPQN G RHKQN DE rious B54 prefer- FWY l° Anchor FWYLIVM LIVM ALIVM FWYAP l° Anchor 01 red P ATIVLMFWY delete- GPQNDE GDESTC RHKDE DE QNDGE DE rious

TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies of HLA-supertypes in different ethnic populations Specificity Phenotypic frequency N.A. Supertype Position 2 C-Terminus Caucasian Black Japanese Chinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 Anesthesia AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 A24 YF FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 (WIVLMT) B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4 B62 QL (IVMP) FWY (MIV) 12.6  4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV) 10.0 25.1  1.6  9.0  5.9 10.3

TABLE IV (G) Calculated population coverage afforded by different HLA-supertype combinations Phenotypic frequency HLA-supertypes Caucasian N.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, 99.5 98.1 100.0 99.5 99.4 99.3 A24, B44 and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2, A3, B7, A24, B44, A1, B27, B62, and B58 Motifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

TABLE V Frequently Occurring Motifs avrg. Name % identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 Nucleic acid-binding protein functions as type transcription factor, nuclear location probable cytochrome_ 68% Cytochrome b(N- membrane bound oxidase, generate superoxide b_N terminal)/b6/petB Ig 19% Immunoglobulin domains are one hundred amino acids long and domain include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta tandem repeats of about 40 residues, each repeat containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub-membranous sites LRR 28% Leucine Rich short sequence motifs involved in protein-protein Repeat interactions Pkinase 23% Protein kinase conserved catalytic core common to both domain serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins Rvt 49% Reverse transcriptase (RNA- dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton Oxidored_q1 32% NADH- membrane associated. Involved in proton Ubiquinone/plastoq translocation across the membrane uinone (complex I), various chains Efhand 24% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha- helical domain Rvp 79% Retroviral aspartyl Aspartyl or acid proteases, centered on a catalytic protease aspartyl residue Collagen 42% Collagen triple extracellular structural proteins involved in helix repeat (20 formation of connective tissue. The sequence copies) consists of the G-X-Y and the polypeptide chains forms a triple helix. Fn3 20% Fibronectin type III Located in the extracellular ligand-binding region domain of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane seven hydrophobic transmembrane regions, with receptor (rhodopsin the N-terminus located extracellularly while the family) C-terminus is cytoplasmic. Signal through G proteins

TABLE VI Motifs and Post-translational Modifications of 282P1G3 N-glycosylation site 87-90 NNSG (SEQ ID NO: 54) 231-234 NDSS (SEQ ID NO: 55) 315-318 NVSY (SEQ ID NO: 56) 410-413 NHTA (SEQ ID NO: 57) 492-495 NGTL (SEQ ID NO: 58) 498-501 NRTT (SEQ ID NO: 59) 529-532 NATK (SEQ ID NO: 60) 578-581 NGTE (SEQ ID NO: 61) 591-594 NLTI (SEQ ID NO: 62) 596-599 NVTL (SEQ ID NO: 63) 641-644 NRSV (SEQ ID NO: 64) 657-660 NISE (SEQ ID NO: 65) 783-786 NHTL (SEQ ID NO: 66) 838-841 NSTL (SEQ ID NO: 67) 961-964 NLTG (SEQ ID NO: 68) 973-976 NDTY (SEQ ID NO: 69) 985-988 NITT (SEQ ID NO: 70) 1000-1003 NATT (SEQ ID NO: 71) 1042-1045 NLTQ (SEQ ID NO: 72) 1071-1074 NDSI (SEQ ID NO: 73) 1213-1216 NGSS (SEQ ID NO: 74) Tyrosine sulfation site 817-831 TLYSGEDYPDTAPVI (SEQ ID NO: 75) 1083-1097 GREYAGLYDDISTQG (SEQ ID NO: 76) 1145-1159 KDETFGEYSDSDEKP (SEQ ID NO: 77) 1176-1190 SADSLVEYGEGDHGL (SEQ ID NO: 78) cAMP- and cGMP-dependent protein kinase phosphorylation site 684-687 KKTT (SEQ ID NO: 79) Pkinase C phosphorylation site 91-93 TFR 112-114 SNK 183-185 SQK 226-228 SLK 245-247 SIK 310-312 TLK 350-352 TKK 377-379 TIK 536-538 SPK 563-565 SLK 637-639 SER 643-645 SVR 766-768 TWK 785-787 TLR 1002-1004 TTK 1044-1046 TQK 1128-1130 SVK 1143-1145 SVK 1163-1165 SLR Casein kinase II phosphorylation site 198-201 SRND (SEQ ID NO: 80) 235-238 SSTE (SEQ ID NO: 81) 260-263 SGSE (SEQ ID NO: 82) 317-320 SYQD (SEQ ID NO: 83) 385-388 SPVD (SEQ ID NO: 84) 500-503 TTEE (SEQ ID NO: 85) 501-504 TEED (SEQ ID NO: 86) 554-557 SKCD (SEQ ID NO: 87) 598-601 TLED (SEQ ID NO: 88) 611-614 TALD (SEQ ID NO: 89) 615-618 SAAD (SEQ ID NO: 90) 623-626 TVLD (SEQ ID NO: 91) 809-812 SGPD (SEQ ID NO: 92) 820-823 SGED (SEQ ID NO: 93) 870-873 SLLD (SEQ ID NO: 94) 1027-1030 TLGE (SEQ ID NO: 95) 1128-1131 SVKE (SEQ ID NO: 96) 1143-1146 SVKD (SEQ ID NO: 97) 1148-1151 TFGE (SEQ ID NO: 98) 1153-1156 SDSD (SEQ ID NO: 99) 1179-1182  SLVE (SEQ ID NO: 100) Tyrosine kinase phosphorylation site 480-487 KPL.EGRRY (SEQ ID NO: 101) N-myristoylation site 116-121 GIAMSE (SEQ ID NO: 102) 240-245 GSKANS (SEQ ID NO: 103) 261-266 GSESSI (SEQ ID NO: 104) 322-327 GNYRCT (SEQ ID NO: 105) 364-369 GILLCE (SEQ ID NO: 106) 424-429 GTILAN (SEQ ID NO: 107) 506-511 GSYSCW (SEQ ID NO: 108) 579-584 GTEDGR (SEQ ID NO: 109) 589-594 GANLTI (SEQ ID NO: 110) 603-608 GIYCCS (SEQ ID NO: 111) 651-656 GADHNS (SEQ ID NO: 112) 888-893 GQRNSG (SEQ ID NO: 113) 893-898 GMVPSL (SEQ ID NO: 114) 960-965 GNLTGY (SEQ ID NO: 115) 1040-1045 GVNLTQ (SEQ ID NO: 116) 1101-1106 GLMCAI (SEQ ID NO: 117) 1124-1129 GGKYSV (SEQ ID NO: 118) 1162-1167 GSLRSL (SEQ ID NO: 119) 1195-1200 GSFIGA (SEQ ID NO: 120) 1199-1204 GAYAGS (SEQ ID NO: 121) 1208-1213 GSVESN (SEQ ID NO: 122) 1214-1219 GSSTAT (SEQ ID NO: 123) Amidation site 483-486 EGRR (SEQ ID NO: 124) 682-685 QGKK (SEQ ID NO: 125)

TABLE VII Search Peptides v.1 ORF: 272-3946 9-mers, 10-mers and 15-mers (SEQ ID NO: 126) MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK   60 GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS  120 EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV  180 YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG  240 SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG  300 RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST  360 GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS  420 NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK  480 PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP  540 RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE  600 DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE  660 YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH  720 ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET  780 VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDYPDTAPV IHGVDVINST  840 LVKVTWSTVP KDRVHGRLKG YQINWWKTKS LLDGRTHPKE VNILRFSGQR NSGMVPSLDA  900 FSEFHLTVLA YNSKGAGPES EPYIFQTPEG VPEQPTFLKV IKVDKDTATL SWGLPKKLNG  960 NLTGYLLQYQ IINDTYEIGE LNDINITTPS KPSWHLSNLN ATTKYKFYLR ACTSQGCGKP 1020 ITEESSTLGE GSKGIGKISG VNLTQKTHPI EVFEPGAEHI VRLMTKNWGD NDSIFQDVIE 1080 TRGREYAGLY DDISTQGWFI GLMCAIALLT LLLLTVCFVK RNRGGKYSVK EKEDLHPDPE 1140 IQSVKDETFG EYSDSDEKPL KGSLRSLNRD MQPTESADSL VEYGEGDHGL FSEDGSFIGA 1200 YAGSKEKGSV ESNGSSTATF PLRA 1224 v.2 ORF: 272-3787 9-mers aa 125-141 FIVPSVPKFPKEKIDPL (SEQ ID NO: 127) aa 295-311 GDLPKGREAKENYGKTL (SEQ ID NO: 128) aa 1024-1040 ESSTLGEGKYAGLYDDI (SEQ ID NO: 129) 10-mers aa 124-142 EFIVPSVPKFPKEKIDPLE (SEQ ID NO: 130) aa 294-312 GGDLPKGREAKENYGKTLK (SEQ ID NO: 131) aa 1023-1041 EESSTLGEGKYAGLYDDIS (SEQ ID NO: 132) 15-mers aa 119-147 MSEEIEFIVPSVPKFPKEKIDPLEVEEGD (SEQ ID NO: 133) aa 289-317 DWNKIGGDLPKGREAKENYGKTLKIENVS (SEQ ID NO: 134) aa 1018-1046 GKPITEESSTLGEGKYAGLYDDISTQGWF (SEQ ID NO: 135) v.3 ORF: 272 . . . 2953 Frame + 2 9-mers aa 830-848 VIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF FKI (SEQ ID NO: 136) 10-mers aa 829-849 PVIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF FKI (SEQ ID NO: 137) 15-mers aa 824-854 YPDTAPVIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF FKI (SEQ ID NO: 138) v.4 ORF: 272 . . . 3625 Frame + 2 9-mers aa 816-832 VTLYSGEDLPEQPTFLK (SEQ ID NO: 139) 10-mers aa 815-833 SVTLYSGEDLPEQPTFLKV (SEQ ID NO: 140) 15-mers aa 810-838 GPDPQSVTLYSGEDLPEQPTFLKVIKVDK (SEQ ID NO: 141) v.5 ORF: 272 . . . 3898 Frame + 2 9-mers aa 219-235 PMKLTVNSSNSIKQRKP (SEQ ID NO: 142) 10-mers aa 218-236 MPMKLTVNSSNSIKQRKPK (SEQ ID NO: 143) 15-mers aa 213-241 TIVQKMPMKLTVNSSNSIKQRKPKLLLPP (SEQ ID NO: 144) v.6 ORF: 272 . . . 3823 Frame + 2 9-mers aa 121-137 EEIEFIVPKLEHIEQDE (SEQ ID NO: 145) 10-mers aa 122-139 SEEIEFIVPKLEHIEQDER (SEQ ID NO: 146) 15-mers aa 115-143 LGIAMSEEIEFIVPKLEHIEQDERVYMSQ (SEQ ID NO: 147) v.7 ORF: 272 . . . 3982 Frame + 2 9-mers aa 337-364 HDFHVIVEDNISHELFTLHPEPPRWTKK (SEQ ID NO: 148) 10 mers aa 336-365 THDFHVIVEDNISHELFTLHPEPPRWTKKP (SEQ ID NO: 149) 15-mers aa 331-370 FLGTATHDFHVIVEDNISHELFTLHPEPPRWTKKPQSAVY (SEQ ID NO: 150)

Tables VIII-XXI:

TABLE VIII V1-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 500 TTEEDAGSY 112.500 919 ESEPYIFQT 67.500 173 HIEQDERVY 45.000 1078 VIETRGREY 45.000 371 EGEPQPTIK 45.000 931 VPEQPTFLK 22.500 524 NLDIRNATK 20.000 760 GLEYRVTWK 18.000 547 MLELHCESK 18.000 579 GTEDGRIII 11.250 871 LLDGRTHPK 10.000 343 VEEPPRWTK 9.000 1191 FSEDGSFIG 6.750 119 MSEEIEFIV 6.750 78 FTDHRIIPS 6.250 145 EGDPIVLPC 6.250 721 ETPPAAPDR 5.000 915 GAGPESEPY 5.000 396 VVFPREISF 5.000 168 NIELEHIEQ 4.500 598 TLEDQGIYC 4.500 917 GPESEPYIF 4.500 149 IVLPCNPPK 4.000 1154 DSDEKPLKG 3.750 434 VVDVRPLIQ 2.500 948 ATLSWGLPK 2.500 961 NLTGYLLQY 2.500 287 QVDWNKIGG 2.500 789 MTPAVYAPY 2.500 586 IIDGANLTI 2.500 810 GPDPQSVTL 2.500 236 STEIGSKAN 2.250 1021 ITEESSTLG 2.250 1183 YGEGDHGLF 2.250 62 NPEPTFSWT 2.250 416 QCEASNVHG 1.800 142 EVEEGDPIV 1.800 122 EIEFIVPSV 1.800 1175 ESADSLVEY 1.500 261 GSESSITIL 1.350 901 FSEFHLTVL 1.350 627 VPDPPENLH 1.250 1144 VKDETFGEY 1.250 1136 HPDPEIQSV 1.250 816 VTLYSGEDY 1.250 70 TKDGNPFYF 1.250 597 VTLEDQGIY 1.250 157 KGLPPLHIY 1.250 571 DGEAFEING 1.125 270 KGEILLLEC 1.125 978 IGELNDINI 1.125 1112 LLLTVCFVK 1.000 137 KIDPLEVEE 1.000 616 AADITQVTV 1.000 45 VAFPFDEYF 1.000 835 DVINSTLVK 1.000 279 FAEGLPTPQ 0.900 369 EAEGEPQPT 0.900 54 QIECEAKGN 0.900 342 IVEEPPRWT 0.900 192 NVEEKDSRN 0.900 753 SMEQNGPGL 0.900 511 WVENAIGKT 0.900 1056 GAEHIVRLM 0.900 551 HCESKCDSH 0.900 367 LCEAEGEPQ 0.900 1180 LVEYGEGDH 0.900 275 LLECFAEGL 0.900 24 AIEIPSSVQ 0.900 1209 SVESNGSST 0.900 738 ASQPKEMII 0.750 316 VSYSDEKPL 0.750 1152 YSDSDEKPL 0.750 199 RNDYCCFAA 0.625 1068 WGDNDSIFQ 0.625 44 QVAFPFDEY 0.500 99 HISHFQDEY 0.500 1000 NATTKYKFY 0.500 158 GLPPLHIYW 0.500 117 IAMSEEIEF 0.500 392 FAGDVVFPR 0.500 1176 SADSLVEYG 0.500 612 ALDSAADIT 0.500 651 GADHNSNIS 0.500 875 RTHPKEVNI 0.500 833 GVDVINSTL 0.500 202 YCCFAAFPR 0.500 897 SLDAFSEFH 0.500 906 LTVLAYNSK 0.500 986 ITTPSKPSW 0.500 555 KCDSHLKHS 0.500 893 GMVPSLDAF 0.500 957 KLNGNLTGY 0.500 689 ILPLAPFVR 0.500 853 RVHGRLKGY 0.500 13 YLMFLLLKF 0.500 929 EGVPEQPTF 0.500 1052 VFEPGAEHI 0.450 213 TIVQKMPMK 0.400 949 TLSWGLPKK 0.400 V2-HLA-Al- 9 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 FIVPSVPKF 2.000 3 VPSVPKFPK 0.250 5 SVPKFPKEK 0.020 4 PSVPKFPKE 0.003 2 IVPSVPKFP 0.001 6 VPKFPKEKI 0.000 9 FPKEKIDPL 0.000 7 PKFPKEKID 0.000 8 KFPKEKIDP 0.000 V2-HLA-Al- 9 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 AKENYGKTL 0.045 6 GREAKENYG 0.045 2 DLPKGREAK 0.020 5 KGREAKENY 0.013 1 GDLPKGREA 0.005 7 REAKENYGK 0.002 8 EAKENYGKT 0.001 3 LPKGREAKE 0.000 4 PKGREAKEN 0.000 V2-HLA-Al- 9 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 SSTLGEGKY 0.750 5 LGEGKYAGL 0.450 1 ESSTLGEGK 0.300 3 STLGEGKYA 0.025 4 TLGEGKYAG 0.020 6 GEGKYAGLY 0.003 9 KYAGLYDDI 0.001 7 EGKYAGLYD 0.000 8 GKYAGLYDD 0.000 V3-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 4 GVDVINTTY 25.000 36 EQELSYRNR 1.350 10 TTYVSNTTY 1.250 55 STSCNYVEK 1.000 46 MLAEDFIQK 1.000 47 LAEDFIQKS 0.900 60 YVEKSSTFF 0.900 34 SKEQELSYR 0.450 33 CSKEQELSY 0.375 23 TGSPQPSIF 0.250 25 SPQPSIFIC 0.125 24 GSPQPSIFI 0.075 22 ATGSPQPSI 0.050 27 QPSIFICSK 0.050 16 TTYVSNATG 0.050 13 VSNTTYVSN 0.030 15 NTTYVSNAT 0.025 48 AEDFIQKST 0.025 45 NMLAEDFIQ 0.025 9 NTTYVSNTT 0.025 1 VIHTTYVSN 0.020 7 VINTTYVSN 0.020 12 YVSNTTYVS 0.020 6 DVINTTYVS 0.020 19 VSNATGSPQ 0.015 56 TSCNYVEKS 0.015 29 SIFICSKEQ 0.010 21 NATGSPQPS 0.010 57 SCNYVEKSS 0.010 38 ELSYRNRNM 0.010 31 FICSKEQEL 0.010 52 IQKSTSCNY 0.007 61 VEKSSTFFK 0.005 59 NYVEKSSTF 0.005 43 NRNMLAEDF 0.005 54 KSTSCNYVE 0.003 3 HGVDVINTT 0.003 44 RNMLAEDFI 0.003 8 INTTYVSNT 0.003 58 CNYVEKSST 0.003 14 SNTTYVSNA 0.003 62 EKSSTFFKI 0.003 2 IHGVDVINT 0.003 39 LSYRNRNML 0.002 51 FIQKSTSCN 0.001 18 YVSNATGSP 0.001 32 ICSKEQELS 0.001 35 KEQELSYRN 0.001 26 PQPSIFICS 0.001 37 QELSYRNRN 0.001 20 SNATGSPQP 0.001 5 VDVINTTYV 0.001 49 EDFIQKSTS 0.001 11 TYVSNTTYV 0.001 50 DFIQKSTSC 0.001 53 QKSTSCNYV 0.001 17 TYVSNATGS 0.001 40 SYRNRNMLA 0.000 28 PSIFICSKE 0.000 42 RNRNMLAED 0.000 30 IFICSKEQE 0.000 41 YRNRNMLAE 0.000 V4-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 LPEQPTFLK 22.500 7 EDLPEQPTF 0.100 4 YSGEDLPEQ 0.030 6 GEDLPEQPT 0.025 1 VTYLSGEDL 0.025 5 SGEWLPEQP 0.022 8 DLPEQPTFL 0.010 2 TLYSGEDLP 0.001 3 LYSGEDLPE 0.000 V5-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 8 SSNSIKQRK 0.300 5 TVNSSNSIK 0.200 7 NSSNSIKQR 0.150 4 LTVNSSNSI 0.025 6 VNSSNSIKQ 0.013 3 KLTVNSSNS 0.010 2 MKLTVNSSN 0.001 9 SNSIKQRKP 0.000 1 PMKLTVNSS 0.000 V6-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 EIEFIVPKL 1.800 5 FIVPKLEHI 0.100 9 KLEHIEQDE 0.090 1 EEIEFIVPK 0.020 4 EFIVPKLEH 0.003 7 VPKLEHIEQ 0.001 6 IVPKLEHIE 0.000 3 IEFIVPKLE 0.000 8 PKLEHIEQD 0.000 V7-HLA-Al- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 19 HPEPPRWTK 45.000 16 FTLHPEPPR 0.500 17 TLHPEPPRW 0.200 6 IVEDNISHE 0.090 5 VIVEDNISH 0.050 10 NISHELFTL 0.050 7 VEDNISHEL 0.025 12 SHELFTLHP 0.022 11 ISHELFTLH 0.015 9 DNISHELFT 0.013 20 PEPPRWTKK 0.010 4 HVIVEDNIS 0.010 8 EDNISHELF 0.005 14 ELFTLHPEP 0.002 2 DFHVIVEDN 0.001 18 LHPEPPRWT 0.001 3 FHVIVEDNI 0.001 1 HDFHVIVED 0.000 15 LFTLHPEPP 0.000 13 HELFTLHPE 0.000

TABLE IX V1-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 261 GSESSITILK 135.000 810 GPDPQSVTLY 62.500 62 NPEPTFSWTK 45.000 1152 YSDSDEKPLK 30.000 1136 HPDPEIQSVK 25.000 1028 LGEGSKGIGK 22.500 312 KIENVSYQDK 18.000 342 IVEEPPRWTK 18.000 738 ASQPKEMIIK 15.000 371 EGEPQPTIKW 11.250 406 NLQPNHTAVY 10.000 343 VEEPPRWTKK 9.000 170 ELEHIEQDER 9.000 658 ISEYIVEFEG 6.750 1191 FSEDGSFIGA 6.750 627 VPDPPENLHL 6.250 788 VMTPAVYAPY 5.000 688 VILPLAPFVR 5.000 137 KIDPLEVEEG 5.000 1056 GAEHIVRLMT 4.500 481 PLEGRRYHIY 4.500 236 STEIGSKANS 4.500 1149 FGEYSDSDEK 4.500 466 SPEAVVSWQK 4.500 475 KVEEVKPLEG 4.500 142 EVEEGDPIVL 4.500 901 FSEFHLTVLA 2.700 434 VVDVRPLIQT 2.500 897 SLDAFSEFHL 2.500 78 FTDHRIIPSN 2.500 145 EGDPIVLPCN 2.500 4 LLLGRGLIVY 2.500 199 RNDYCCFAAF 2.500 612 ALDSAADITQ 2.500 500 TTEEDAGSYS 2.250 747 KWEPLKSMEQ 2.250 270 KGEILLLECF 2.250 369 EAEGEPQPTI 1.800 279 FAEGLPTPQV 1.800 460 HCEFFASPEA 1.800 173 HIEQDERVYM 1.800 24 AIEIPSSVQQ 1.800 300 GRETKENYGK 1.800 598 TLEDQGIYCC 1.800 919 ESEPYIFQTP 1.350 636 LSERQNRSVR 1.350 1192 SEDGSFIGAY 1.250 499 RTTEEDAGSY 1.250 917 GPESEPYIFQ 1.125 1021 ITEESSTLGE 1.125 1183 YGEGDHGLFS 1.125 579 GTEDGRIIID 1.125 931 VPEQPTFLKV 1.125 930 GVPEQPTFLK 1.000 948 ALTSWGLPKK 1.000 1111 LLLLTVCFVK 1.000 212 RTIVQKMPMK 1.000 11 IVYLMFLLLK 1.000 126 IVPLAPFVRY 1.000 689 ILPLAPFVRY 1.000 30 SVQQVPTIIK 1.000 624 VLDVPDPPEN 1.000 947 TATLSWGLPK 1.000 1078 VIETRGREYA 0.900 511 WVENAIGKTA 0.900 1180 LVEYGEGDHG 0.900 416 QCEASNVHGT 0.900 547 MLELHCESKC 0.900 551 HCESKCDSHL 0.900 303 TKENYGKTLK 0.900 675 WEELTRVQGK 0.900 1209 SVESNGSSTA 0.900 996 LSNLNATTKY 0.750 1154 DSDEKPLKGS 0.750 1075 FQDVIETRGR 0.750 119 MSEEIEFIVP 0.675 824 YPDTAPVIHG 0.625 960 GNLTGYLLQY 0.625 431 NIDVVDVRPL 0.500 616 AADITQVTVL 0.500 586 IIDGANLTIS 0.500 847 STVPKDRVHG 0.500 395 DVVFPREISF 0.500 524 NLDIRNATKL 0.500 555 KCDSHLKHSL 0.500 833 GVDVINSTLV 0.500 686 TTVILPLAPF 0.500 596 NVTLEDQGIY 0.500 14 LMFLLLKFSK 0.500 315 NVSYQDKGNY 0.500 815 SVTLYSGEDY 0.500 478 EVKPLEGRRY 0.500 440 LIQTKDGENY 0.500 283 LPTPQVDWNK 0.500 116 GIAMSEEIEF 0.500 1077 DVIETRGREY 0.500 1109 LTLLLLTVCF 0.500 1134 DLHPDPEIQS 0.500 445 DGENYATVVG 0.450 669 KEEPGRWEEL 0.450 V2-HLA-Al- 10 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 3 IVPSVPKFPK 1.000 5 PSVPKFPKEK 0.300 1 EFIVPSVPKF 0.010 2 FIVPSVPKFP 0.010 6 SVPKFPKEKI 0.001 4 VPSVPKFPKE 0.001 8 PKFPKEKIDP 0.000 10 FPKEKIDPLE 0.000 9 KFPKEKIDPL 0.000 7 VPKFPKEKID 0.000 V2-HLA-Al- 10 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 LGEGKYAGLY 11.250 1 ESSTLGEHKY 0.750 3 STLGEGKYAG 0.050 4 TLGEGKYAGL 0.020 2 SSTLGEGKYA 0.015 8 GKYAGLYDDI 0.001 7 EGKYAGLYDD 0.000 6 GEGKYAGLYD 0.000 V2-HLA-Al- 10 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 6 LGEGKYAGLY 11.250 2 ESSTLGEHKY 0.750 4 STLGEGKYAG 0.050 5 TLGEGKYAGL 0.020 3 SSTLGEGKYA 0.015 1 EESSTLGECK 0.010 10 KYAGLYDDIS 0.001 9 GKYAGLTDDI 0.001 8 EGKYAGLYDD 0.000 7 GEGKYAGLYD 0.000 V3-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 61 YVEKSSTFFK 9.000 10 NTTYVSNTTY 1.250 48 LAEDFIQKST 0.900 55 KSTSCNYVEK 0.600 46 NMLAEDFIQK 0.500 5 GVDVINTTYV 0.500 33 ICSKEQELSY 0.250 23 ATGSPQPSIF 0.250 37 EQELSYRNRN 0.135 26 SPQPSIFICS 0.125 4 HGVDVINTTY 0.125 24 TGSPQPSIFI 0.125 35 SKEQELSYRN 0.090 25 GSPQPSIFIC 0.075 52 FIQKSTSCNY 0.050 16 NTTYVSNATG 0.050 2 VIHGVDVINT 0.050 49 AEDFIQKSTS 0.025 56 STSCNYVEKS 0.025 11 TTYVSNTTYV 0.025 17 TTYVSNATGS 0.025 59 CNYVEKSSTF 0.025 13 YVSNTTYVSN 0.020 22 NATGSPQPSI 0.020 7 DVINTTYVSN 0.020 34 CSKEQELSYR 0.015 14 VSNTTYVSNA 0.015 57 TSCNYVEKSS 0.015 45 RNMLAEDFIQ 0.013 47 MLAEDFIQKS 0.010 32 FICSKEQELS 0.010 58 SCNYVEKSST 0.010 8 VINTTYVSNT 0.010 19 YVSNATGSPQ 0.010 39 ELSYRNRNML 0.010 40 LSYRNRNMLA 0.008 60 NYVEKSSTFF 0.005 36 KEQELSYRNR 0.005 20 VSNATGSPQP 0.003 27 PQPSIFICSK 0.003 15 SNTTYVSNAT 0.003 43 RNRNMLAEDF 0.003 9 INTTYVSNTT 0.003 21 SNATGSPQPS 0.003 1 PVIHGVDVIN 0.002 29 PSIFICSKEQ 0.002 12 TYVSNTTYVS 0.001 30 SIFICSKEQE 0.001 6 VDVINTTYVS 0.001 31 IFICSKEQEL 0.001 38 QELSYRNRNM 0.001 3 IHGVDVINTT 0.001 51 DFIQKSTSCN 0.001 50 EDFIQKSTSC 0.001 44 NRNMLAEDFI 0.001 62 VEKSSTFFKI 0.000 28 QPSIFICSKE 0.000 53 IQKSTSCNYV 0.000 54 QKSTSCNYVE 0.000 18 TYVSNATGSP 0.000 41 SYRNRNMLAE 0.000 42 YRNRNMLAED 0.000 V4-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 LPEQPTFLKV 1.125 9 DLPEQPTFLK 1.000 7 GEDLPEQPTF 0.500 6 SGEDLPEQPT 0.225 1 SVTLYSGEDL 0.010 8 EDLPEQPTFL 0.005 3 TLYSGEDLPE 0.005 2 VTLYSGEDLP 0.003 5 YSGEDLPEQP 0.002 4 LYSGEDLPEQ 0.001 V5-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 LTVNSSNSIK 0.500 8 NSSNSIKQRK 0.300 10 SNSIKQRKPK 0.050 6 TVNSSNSIKQ 0.050 7 VNSSNSIKQR 0.025 4 KLTVNSSNSI 0.010 9 SSNSIKQRKP 0.002 3 MKLTVNSSNS 0.001 1 MPMKLTVNSS 0.000 2 PMKLTVNSSN 0.000 V6-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 KLEHIEQDER 9.000 1 SEEIEFIVPK 1.800 3 EIEFIVPKLE 0.090 6 FIVPKLEHIE 0.010 7 IVPKLEHIEQ 0.005 4 IEFIVPKLEH 0.003 2 EEIEFIVPKL 0.001 5 EFIVPKLEHI 0.001 8 VPKLEHIEQD 0.000 9 PKLEHIEQDE 0.000 V7-HLA-Al- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 20 HPEPPRWTKK 45.000 7 IVEDNISHEL 0.900 8 VEDNISHELF 0.250 18 TLHPEPPRWT 0.100 5 HVIVEDNISH 0.050 17 FTLHPEPPRW 0.050 10 DNISHELFTL 0.013 19 LHPEPPRWTK 0.010 16 LFTLHPEPPR 0.010 11 NISHELFTLH 0.010 12 ISHELFTLHP 0.007 1 THDFHVIVED 0.005 13 SHELFTLHPE 0.005 9 EDNISHELFT 0.003 15 ELFTLHPEPP 0.001 6 VIVEDNISHE 0.001 2 HDFHVIVEDN 0.001 4 FHVIVEDNIS 0.001 3 DFHVIVEDNI 0.001 14 HELFTLHPEP 0.000 21 PEPPRWTKKP 0.000

TABLE X V1-HLA-Al- A0201-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1111 LLLLTVCFV 5534.148 688 VILPLAPFV 330.846 1108 LLTLLLLTV 271.948 9 GLIVYLMFL 270.234 17 LLLKFSKAI 249.365 118 AMSEEIEFI 191.488 1101 GLMCAIALL 181.794 4 LLLGRGLIV 179.368 16 FLLLKFSKA 160.655 426 ILANANIDV 118.238 923 YIFQTPEGV 79.757 406 NLQPNHTAV 69.552 10 LIVYLMFLL 66.613 1107 ALLTLLLLT 63.417 840 TLVKVTWST 55.890 1027 TLGEGSKGI 42.774 930 GVPEQPTFL 42.151 47 FPFDEYFQI 41.346 166 WMNIELEHI 39.062 836 VINSTLVKV 37.393 125 FIVPSVPKL 31.077 591 NLTISNVTL 21.362 11 IVYLMFLLL 19.320 544 KLHMLELHC 17.388 1166 SLNRDMQPT 17.140 23 KAIEIPSSV 13.862 103 FQGKYRCFA 12.744 267 TILKGEILL 10.868 1042 NLTQKTHPI 10.433 451 TVVGYSAFL 10.281 1001 ATTKYKFYL 9.465 967 LQYQIINDT 9.453 1102 LMCAIALLT 9.149 787 RVMTPAVYA 8.846 1073 SIFQDVIET 8.720 908 VLAYNSKGA 8.446 471 VSWQKVEEV 7.220 598 TLEDQGIYC 7.170 585 IIIDGANLT 7.142 335 ATHDFHVIV 6.171 680 RVQGKKTTV 6.086 780 TVTNHTLRV 6.086 333 GTATHDFHV 5.603 785 TLRVMTPAV 5.286 1092 DISTQGWFI 4.438 275 LLECFAEGL 4.328 1106 IALLTLLLL 4.292 14 LMFMMMKFS 4.282 458 FLHCEFFAS 3.778 619 ITQVTVLDV 3.777 174 IEQDERVYM 3.703 1033 KGIGKISGV 3.655 274 LLLECFAEG 3.651 774 VEWEEETVT 3.437 603 GIYCCSAHT 3.279 214 IVQKMPMKL 3.178 1105 AIALLTLLL 2.937 584 RIIIDGANL 2.937 268 ILKGEILLL 2.923 13 YLMFLLLKF 2.917 942 KVDKDTATL 2.617 1053 FEPGAEHIV 2.551 980 ELNDINITT 2.291 939 KVIKVDKDT 2.282 429 NANIDVVDV 2.222 589 GANLTISNV 2.222 611 TALDSAADI 2.198 976 YEIGELNDI 2.146 444 KDGENYATV 2.079 863 INWWKTKSL 1.968 950 LSWGLPKKL 1.968 83 IIPSNNSGT 1.742 26 EIPSSVQQV 1.650 916 AGPESEPYI 1.536 970 QIINDTYEI 1.435 819 YSGEDYPDT 1.376 280 AEGLPTPQV 1.352 1099 FIGLMCAIA 1.288 185 KGDLYFANV 1.208 375 PQPTIKWRV 1.164 991 KPSWHLSNL 1.123 988 TPSKPSWHL 1.046 272 EILLLECFA 1.043 846 WSTVPKDRV 1.023 753 SMEQNGPGL 0.987 203 CCFAAFPRL 0.980 586 IIDGANLTI 0.975 1066 KNWGDNDSI 0.969 252 KLLLPPTES 0.965 743 EMIIKWEPL 0.964 318 YQDKGNYRC 0.927 746 IKWEPLKSM 0.918 534 RSVPKNPRI 0.913 596 NVTLEDQDI 0.913 1100 IGLMCAIAL 0.877 210 RLRTIVQKM 0.868 736 VQASQPKEM 0.856 370 AEGEPQPTI 0.832 1214 GSSTATFPL 0.809 427 LANANIDVV 0.759 V2-HLA-A201- 9 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 FIVPSVPKF 2.000 3 VPSVPKFPK 0.250 5 SVPKFPKEK 0.020 4 PSVPKFPKE 0.003 2 IVPSVPKFP 0.001 6 VPKFPKEKI 0.000 9 FPKEKIDPL 0.000 7 PKFPKEKID 0.000 8 KFPKEKIDP 0.000 V2-HLA-A201- 9 mers-(SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 GDLPKGREA 0.005 9 AKENYGKTL 0.002 2 DLPKGREAK 0.001 7 REAKENYGK 0.000 5 KGREAKENY 0.000 8 EAKENYGKT 0.000 3 LPKGREAKE 0.000 6 GREAKENYG 0.000 4 PKGREAKEN 0.000 V2-HLA-A201- 9 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 3 STLGEGKYA 1.404 4 TLGEGKYAG 0.306 5 LGEGKYAGL 0.023 9 KYAGLYDDI 0.004 6 GEGKYAGLY 0.000 8 GKYAGLYDD 0.000 2 SSTLGEGKY 0.000 1 ESSTLGEGK 0.000 7 EGKYAGLYD 0.000 V3-HLA-A201- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 31 FICSKEQEL 13.512 5 VDVINTTYV 0.903 39 LSYRNRNML 0.759 44 RNMLAEDFI 0.679 53 QKSTSCNYV 0.531 24 GSPQPSIFI 0.375 46 MLAEDFIQK 0.197 8 INTTYVSNT 0.190 25 SPQPSIFIC 0.177 58 CNYVEKSST 0.156 22 ATGSPQPSI 0.145 9 NTTYVSNTT 0.104 15 NTTYVSNAT 0.104 45 NMLAEDFIQ 0.095 14 SNTTYVSNA 0.075 38 ELSYRNRNM 0.075 48 AEDFIQKST 0.058 11 TYVSNTTYV 0.053 51 FIQKSTSCN 0.047 7 VINTTYVSN 0.026 35 KEQELSYRN 0.021 2 IHGVDVINT 0.020 3 HGVDVINTT 0.016 12 YVSNTTYVS 0.012 62 EKSSTFFKI 0.012 60 YVEKSSTFF 0.011 29 SIFICSKEQ 0.008 1 VIHGVDVIN 0.007 37 QELSYRNRN 0.005 47 LAEDFIQKS 0.004 10 TTYVSNTTY 0.003 16 TTYVSNATG 0.003 4 GVDVINTTY 0.003 13 VSNTTYVSN 0.001 21 NATGSPQPS 0.001 27 QPSIFICSK 0.001 18 YVSNATGSP 0.001 61 VEKSSTFFK 0.001 56 TSCNYVEKS 0.001 57 SCNYVEKSS 0.000 52 IQKSTSCNY 0.000 32 ICSKEQELS 0.000 26 PQPSIFICS 0.000 55 STSCNYVEK 0.000 50 DFIQKSTSC 0.000 6 DVINTTYVS 0.000 23 TGSPQPSIF 0.000 19 VSNATGSPQ 0.000 54 KSTSCNYVE 0.000 20 SNATGSPQP 0.000 33 CSKEQELSY 0.000 40 SYRNRNMLA 0.000 59 NYVEKSSTF 0.000 49 EDFIQKSTS 0.000 41 YRNRNMLAE 0.000 42 RNRNMLAED 0.000 34 SKEQELSYR 0.000 17 TYVSNATGS 0.000 30 IFICSKEQE 0.000 43 NRNMLAEDF 0.000 36 EQELYRNR 0.000 28 PSIFICSKE 0.000 V4-HLA-A0201- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 8 DLPEQPTFL 36.129 1 VTLYSGEDL 0.914 6 GEDLPEQPT 0.058 2 TLYSGEDLP 0.023 4 YSGEDLPEQ 0.004 9 LPEQPTFLK 0.000 7 EDLPEQPTF 0.000 5 SGEDLPEQP 0.000 3 LYSGEDLPE 0.000 V5-HLA-A0201- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 3 KLTVNSSNS 0.261 4 LTVNSSNSI 0.246 2 MKLTVNSSN 0.001 5 TVNSSNSIK 0.001 7 NSSNSIKQR 0.000 6 VNSSNSIKQ 0.000 8 SSNSIKQRK 0.000 1 PMKLTVNSS 0.000 9 SNSIKQRKP 0.000 V6-HLA-A0201- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 FIVPKLEHI 7.437 2 EIEFIVPKL 0.032 9 KLEHIEQDE 0.003 3 IEFIVPKLE 0.002 6 IVPKLEHIE 0.001 1 EEIEFIVPK 0.001 8 PKLEHIEQD 0.000 7 VPKLEHIEG 0.000 4 EFIVPLEH 0.000 V7-HLA-A0201- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 10 NISHELFTL 39.184 7 VEDNISHEL 0.282 17 TLHPEPPRW 0.075 5 VIVEDNISH 0.071 18 LHPEPPRWT 0.040 9 DNISHELFT 0.020 3 FHVIVEDNI 0.016 11 ISHELFTLH 0.006 14 ELFTLHPEP 0.004 16 FTLHPEPPR 0.004 6 IVEDNISHE 0.001 4 HVIVEDNIS 0.000 13 HELFTLHPE 0.000 20 PEPPRWTKK 0.000 15 LFTLHPEPP 0.000 1 HDFHVIVED 0.000 2 DFHVIVEDN 0.000 8 EDNIDHELF 0.000 12 SHELFTLHP 0.000 19 HPEPPRWTK 0.000

TABLE XI V1-HLA- A0201-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the lenght of peptide is 10 amino acids, and the end position for each peptide is the starte position plus nine. Start Subsequence Score 1110 TLLLLTVCFV 3255.381 274 LLLECFAEGL 1025.804 l6 FLLLKFSKAI 674.752 1107 ALLTLLLLTV 591.888 118 AMSEEIEFIV 489.752 5 LLGRGLIVYL 459.398 9 GLIVYLMFLL 284.974 1189 GLFSEDGSFI 212.307 840 TLVKVTWSTV 118.238 132 KLPKEKIDPL 84.264 158 GLPPLHIYWM 62.845 1102 LMCAIALLTL 60.325 426 ILANANIDVV 54.634 957 KLNGNLTGYL 53.459 396 VVFPREISFT 51.883 897 SLDAFSEFHL 49.561 221 KLTVNSLKHA 39.992 150 VLPCNPPKGL 36.316 425 TILANANIDV 35.385 687 TVILPLAPFV 33.472 966 LLQYQIINDT 29.137 1101 GLMCAIALLT 27.572 267 TILKGEILLL 24.997 949 TLSWGLPKKL 21.362 792 AVYAPYDVKV 19.475 413 AVYQCEASNV 19.475 114 KLGIAMSEEI 17.892 13 YLMFLLLKFS 16.044 765 VTWKPQGAPV 13.630 1099 FIGLMCAIAL 13.512 470 VVSWQKVEEV 11.660 585 IIIDGANLTI 9.999 597 VTLEDQGIYC 9.787 693 APFVRYQFRV 9.743 36 TIIKQSKVQV 9.563 10 LIVYLMFLLL 9.488 1000 NATTKYKFYL 9.465 524 NLDIRNATKL 8.545 456 SAFLHCEFFA 8.144 1108 LLTLLLLTVC 7.964 341 VIVEEPPRWT 7.856 752 KSMEQNGPGL 7.404 859 KGYQINWWKT 6.947 541 RPKLHMLEL 6.756 1105 AIALLTLLLL 6.756 8 RGLIVYLMFL 6.527 117 IAMSEEIEFI 5.649 25 IEIPSSVQQV 5.288 969 YQIINDTYEI 4.866 441 IQTKDGENYA 4.710 742 KEMIIKWEPL 4.481 332 LGTATHDFHV 4.477 615 SAADITQVTV 3.961 141 LEVEEGDPIV 3.865 480 KPLEGRRYHI 3.616 598 TLEDQGIYCC 2.998 1034 GIGKISGVNL 2.937 213 TIVQKMPMKL 2.937 862 QINWWKTKSL 2.937 356 AVYSTGSNGI 2.921 839 STLVKVTWST 2.872 953 GLPKKLNGNL 2.777 214 IVQKMPMKLT 2.550 461 CEFFASPEAV 2.452 833 GVCVINSTLV 2.434 3 PLLLGRGLIV 2.321 512 VENAIGKTAV 2.299 565 KLSWSKDGEA 2.260 1181 VEYGEGDHGL 2.260 987 TTPSKPSWHL 2.225 603 GIYCCSAHTA 2.186 61 GNPEPTFSWT 2.084 795 APYDVKVQAI 2.055 1100 IGLMCAIALL 2.017 218 MPMKLTVNSL 2.017 863 INWWKTKSLL 1.968 635 HLSERQNRSV 1.939 1172 QPTESADSLV 1.861 1171 MQPTESADSL 1.804 1008 YLRACTSQGC 1.737 405 TNLQPNHTAV 1.680 618 DITQVTVLDV 1.650 836 VINSTLVKVT 1.643 1043 LTQKTHPIEV 1.642 450 ATVVGYSAFL 1.632 907 TVLAYNSKGA 1.608 681 VQGKKTTVIL 1.510 334 TATHDFHVIV 1.505 1106 IALLTLLLLT 1.497 206 AAFPRLRTIV 1.465 452 VVGYSAFLHC 1.404 378 IKWRVNGSPV 1.363 181 YMSQKGDLYF 1.362 202 YCCFAAFPRL 1.217 171 LEHIEQDERV 1.127 1113 LLTVCFVKRN 1.107 835 DVINSTLVKV 1.050 934 QPTFLKVIKV 1.044 428 ANANIDVVDV 1.044 82 RIIPSNNSGT 1.025 V2-HLA- A0201-10 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 6 SVPKFPKEKI 0.447 9 KFPKEKIDPL 0.059 2 FIVPSVPKFP 0.052 3 IVPSVPKFPK 0.013 4 VPSVPKFPKE 0.000 10 FPKEKIDPLE 0.000 1 EFIVPSVPKF 0.000 5 PSVPKFPKEK 0.000 7 VPKFPKEKID 0.000 8 PKFPKEKIDP 0.000 V2-HLA-A0201-10 mers- (SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 TLGEGKYAGL 131.379 2 SSTLGEGKYA 0.178 8 GKYAGLYDDI 0.034 3 STLGEGKYAG 0.004 6 GEGKYAGLYD 0.002 5 LGEGKYAGLY 0.000 1 ESSTLGEGKY 0.000 7 EGKYAGLYDD 0.000 V2-HLA-A0201-10 mers- (SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 TLGEGKYAGL 131.379 3 SSTLGEGKYA 0.178 9 GKYAGLYDDI 0.034 4 STLGEGKYAG 0.004 7 GEGKYAGLYD 0.002 1 EESSTLGEGK 0.000 10 KYAGLYDDIS 0.000 6 LGEGKYAGLY 0.000 2 ESSTLGEGKY 0.000 8 EGKYAGLYDD 0.000 V3-HLA-A0201- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 11 TTYVSNTTYV 17.002 5 GVDVINTTYV 13.389 47 MLAEDFIQKS 4.540 8 VINTTYVSNT 4.006 2 VIHGVDVINT 4.006 53 IQKSTSCNYV 2.308 39 3LSYRNRNML 1.602 24 TGSPQPSIFI 0.375 25 GSPQPSIFIC 0.177 40 LSYRNRNMLA 0.176 22 NATGSPQPSI 0.145 62 VEKSSTFFKI 0.133 14 VSNTTYVSNA 0.127 9 INTTYVSNTT 0.083 46 NMLAEDFIQK 0.076 38 QELSYRNRNM 0.071 15 SNTTYVSNAT 0.049 58 SCNYVEKSST 0.049 52 FIQKSTSCNY 0.047 48 LAEDFIQKST 0.046 13 YVSNTTYVSN 0.045 31 IFICSKEQEL 0.025 32 FICSKEQELS 0.023 3 IHGVDVINTT 0.020 61 YVEKSSTFFK 0.012 19 YVSNATGSPQ 0.006 44 NRNMLAEDFI 0.004 30 SIFICSKEQE 0.004 17 TTYVSNATGS 0.003 50 EDFIQKSTSC 0.002 59 CNYVEKSSTF 0.002 36 KEQELSYRNR 0.001 56 STSCNYVEKS 0.001 10 NTTYVSNTTY 0.001 16 NTTYVSNATG 0.001 26 SPQPSIFICS 0.001 45 RNMLAEDFIQ 0.001 33 ICSKEQELSY 0.001 7 DVINTTYVSN 0.001 49 AEDFIQKSTS 0.001 55 KSTSCNYVEK 0.001 57 TSCNYVEKSS 0.000 21 SNATGSPQPS 0.000 23 ATGSPQPSIF 0.000 27 PQPSIFICSK 0.000 60 NYVEKSSTFF 0.000 34 CSKEQELSYR 0.000 20 VSNATGSPQP 0.000 28 QPSIFICSKE 0.000 6 VDVINTTYVS 0.000 4 HGVDVINTTY 0.000 1 PVIHGVDVIN 0.000 37 EQELSYRNRN 0.000 42 YRNRNMLAED 0.000 43 RNRNMLAEDF 0.000 54 QKSTSCNYVE 0.000 35 SKEQELSYRN 0.000 12 TYVSNTTYVS 0.000 51 DFIQKSTSCN 0.000 29 PSIFICSKEQ 0.000 41 SYRNRNMLAE 0.000 18 TYVSNATGSP 0.000 V4-HLA-0201- 10 mers-282P1G3 Each peptdie is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 SVTLYSGEDL 0.916 10 LPEQPTFLKV 0.094 3 TLYSGEDLPE 0.048 8 EDLPEQPTFL 0.045 9 DLPEQPTFLK 0.027 6 SGEDLPEQPT 0.013 5 YSGEDLPEQP 0.001 2 VTLYSGEDLP 0.001 7 GEDLPEQPTF 0.001 4 LYSGEDLPEQ 0.000 V5-HLA-0201- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 KLTVNSSNSI 36.515 1 MPMKLTVNSS 0.007 6 TVNSSNSIKQ 0.001 3 MKLTVNSSNS 0.001 7 VNSSNSIKQR 0.000 5 LTVNSSNSIK 0.000 10 SNSIKQRKPK 0.000 8 NSSNSIKQRK 0.000 2 PMKLTVNSSN 0.000 9 SSNSIKQRKP 0.000 V6-HLA-0201- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 2 EEIEFIVPKL 0.294 4 IEFIVPKLEH 0.009 6 FIVPKLEHIE 0.004 7 IVPKLEHIEQ 0.002 10 KLEHIEQDER 0.002 5 EFIVPKLEHI 0.001 1 SEEIEFIVPK 0.000 3 EIEFIVPKLE 0.000 9 PKLEHIEQDE 0.000 8 VPKLEHIEQD 0.000 V7-HLA-0201- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 18 TLHPEPPRWT 8.197 7 IVEDNISHEL 0.834 10 DNISHELFTL 0.140 6 VIVEDNISHE 0.033 11 NISHELFTLH 0.019 17 FTLHPEPPRW 0.018 9 EDNISHELFT 0.004 12 ISHELFTLHP 0.003 15 ELFTLHPEPP 0.002 19 LHPEPPRWTK 0.001 8 VEDNISHELF 0.000 3 DFHVIVEDNI 0.000 5 HVIVEDNISH 0.000 4 FHVIVEDNIS 0.000 14 HELFTLHPEP 0.000 16 LFTLHPEPPR 0.000 2 HDFHVIVEDN 0.000 1 THDFHVIVED 0.000 21 PEPPRWTKKP 0.000 13 SHELFTLHPE 0.000 20 HPEPPRWTKK 0.000

TABLE XII V1-HLA-A3- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 760 GLEYRVTWK 180.000 1112 LLLTVCFVK 135.000 961 NLTGYLLQY 54.000 937 FLKVIKVDK 30.000 949 TLSWGLPKK 30.000 871 LLDGRTHPK 30.000 957 KLNGNLTGY 27.000 9 GLIVYLMFL 24.300 893 GMVPSLDAF 20.250 524 NLDIRNATK 20.000 547 MLELHCESK 20.000 792 AVYAPYDVK 15.000 5 LLGRGLIVY 12.000 1113 LLTVCFVKR 12.000 689 ILPLAPFVR 12.000 998 NLNATTKYK 10.000 744 MIIKWEPLK 9.000 1189 GLFSEDGSF 9.000 13 YLMFLLLKF 9.000 948 ATLSWGLPK 9.000 843 KVTWSTVPK 6.000 296 DLPKGRETK 6.000 213 TIVQKMPMK 4.500 149 IVLPCNPPK 4.500 661 YIVEFEGNK 4.050 1101 GLMCAIALL 4.050 181 YMSQKGDLY 4.000 310 TLKIENVSY 4.000 396 VVFPREISF 3.000 1110 TLLLLTVCF 3.000 530 ATKLRVSPK 3.000 268 ILKGEILLL 2.700 677 ELTRVQGKK 2.700 331 FLGTATHDF 2.000 11 IVYLMFLLL 1.800 275 LLECFAEGL 1.800 857 RLKGYQINW 1.800 739 SQPKEMIIK 1.800 44 QVAFPFDEY 1.800 835 DVINSTLVK 1.800 31 VQQVPTIIK 1.800 158 GLPPLHIYW 1.800 1197 FIGAYAGSK 1.800 1002 TTKYKFYLR 1.800 906 LTVLAYNSK 1.500 1199 GAYAGSKEK 1.500 859 KGYQINWWK 1.350 118 AMSEEIEFI 1.350 17 LLLKFSKAI 1.350 436 DVRPLIQTK 1.350 657 NISEYIVEF 1.350 861 YQINWWKTK 1.350 221 KLTVNSLKH 1.200 544 KLHMLELHC 1.200 129 SVPKLPKEK 1.000 166 WMNIELEHI 0.900 931 VPEQPTFLK 0.900 4 LLLGRGLIV 0.900 1111 LLLLTVCFV 0.900 1150 GFYSDSDFK 0.900 867 KTKSLLDGR 0.900 282 GLPTPQVDW 0.900 210 RLRTIVQKM 0.900 242 KANSIKQRK 0.900 16 FLLLKFSKA 0.900 1094 STQGWFIGL 0.810 840 TLVKVTWST 0.675 687 TVILPLAPF 0.675 520 AVTANLDIR 0.600 1163 HIYWMNIEL 0.600 591 NLTISNVTL 0.600 983 DINITTPSK 0.600 1108 LLTLLLLTV 0.600 1042 NLTQKTHPI 0.600 127 VPSVPKLPK 0.600 897 SLDAFSEFH 0.600 1118 FVKRNRGGK 0.600 74 NPFYFTDHR 0.600 340 HVIVEEPPR 0.600 536 SPKNPRIPK 0.600 753 SMEQNGPGL 0.600 882 NILRFSGQR 0.540 392 FAGDVVFPR 0.540 562 HSLKLSWSK 0.450 284 PTPQVDWNK 0.450 853 RVHGRLKGY 0.450 45 VAFPFDEYF 0.450 1027 TLGEGSKGI 0.450 1088 GLYDDISTQ 0.450 1107 ALLTLLLLT 0.450 125 FIVPSVPKL 0.405 10 LIVYLMFLL 0.405 451 TVVGYSAFL 0.405 343 VEEPPRWTK 0.405 702 VIAVNEVGR 0.400 426 ILANANIDV 0.400 598 TLEDQGIYC 0.400 161 PLHIYWMNI 0.360 99 HISHFQGKY 0.360 458 FLHCEFFAS 0.360 V2-HLA-A3- 9 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 SVPKFPKEK 3.000 1 FIVPSVPKF 1.350 3 VPSVPKFPK 0.900 9 FPKEKIDPL 0.013 6 VPKFPKEKI 0.009 2 IVPSVPKFP 0.002 8 KFPKEKIDP 0.000 4 PSVPKFPKE 0.000 7 PKFPKEKID 0.000 V2-HLA-A3- 9 mers-(SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 DLPKGREAK 6.000 7 REAKENYGK 0.180 5 KGREAKENY 0.018 9 AKENYGKTL 0.001 3 LPKGREAKE 0.000 1 GDLPKGREA 0.000 8 EAKENYGKT 0.000 6 GREAKENYG 0.000 4 PKGREAKEN 0.000 V2-HLA-A3- 9 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 4 TLGEGKYAG 0.090 6 GEGKYAGLY 0.032 1 ESSTLGEGK 0.030 3 STLGEGKYA 0.011 2 SSTLGEGKY 0.006 9 KYAGLYDDI 0.003 8 GKYAGLYDD 0.001 5 LGEGKYAGL 0.001 7 EGKYAGLYD 0.000 V3-A3-9 mers- 282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 46 MLAEDFIQK 180.000 4 GVDVINTTY 1.800 10 TTYVSNTTY 1.000 55 STSCNYVEK 1.000 27 QPSIFICSK 0.900 60 YVEKSSTFF 0.200 61 VEKSSTFFK 0.180 52 IQKSTSCNY 0.120 45 NMLAEDFIQ 0.090 33 CSKEQELSY 0.060 31 FICSKEQEL 0.060 22 ATGSPQPSI 0.045 24 GSPQPSIFI 0.027 39 LSYRNRNML 0.015 25 SPQPSIFIC 0.013 12 YVSNTTYVS 0.012 15 NTTYVSNAT 0.007 9 NTTYVSNTT 0.007 34 SKEQELSYR 0.006 38 ELSYRNRNM 0.006 6 DVINTTYVS 0.005 29 SIFICSKEQ 0.005 16 TTYVSNATG 0.005 59 NYVEKSSTF 0.005 1 VIHGVDVIN 0.005 14 SNTTYVSNA 0.004 36 EQELSYRNR 0.004 23 TGSPQPSIF 0.003 43 NRNMLAEDF 0.002 51 FIQKSTSCN 0.002 7 VINTTYVSN 0.002 44 RNMLAEDFI 0.002 8 INTTYVSNT 0.002 56 TSCNYVEKS 0.002 47 LAEDFIQKS 0.002 62 EKSSTFFKI 0.002 26 PQPSIFICS 0.001 58 CNYVEKSST 0.001 54 KSTSCNYVE 0.001 35 KEQELSYRN 0.001 21 NATGSPQPS 0.001 18 YVSNATGSP 0.001 2 IHGVDVINT 0.001 32 ICSKEQELS 0.000 40  SYRNRNMLA 0.000 3 HGVDVINTT 0.000 5 VDVINTTYV 0.000 11 TYVSNTTYV 0.000 57 SCNYVEKSS 0.000 37 WELSYRNRN 0.000 48 AEDFIQKST 0.000 53 QKSTSCNYV 0.000 19 VSNATGSPQ 0.000 13 VSNTTYVSN 0.000 50 DFIQKSTSC 0.000 42 RNRNMLAED 0.000 17 TYVSNATGS 0.000 41 YRNRNMLAE 0.000 49 EDFIQKSTS 0.000 20 SNATGSPQP 0.000 30 IFICSKEQE 0.000 28 PSIFICSKE 0.000 V4-HLA-a3 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 LPEQPTFLK 0.900 8 DLPEQPTFL 0.270 2 TLYSGEDLP 0.100 1 VTLYSGEDL 0.045 7 EDLPEQPTF 0.001 6 DEGLPEQPT 0.001 4 YSGEDLPEQ 0.000 3 LYSGEDLPE 0.000 5 SGEDLPEQP 0.000 V5-HLA-A3- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 TVNSSNSIK 2.000 8 SSNSIKQRK 0.150 3 KLTVNSSNS 0.120 4 LTVNSSNSI 0.045 7 NSSNSIKQR 0.015 1 PMKLTVNSS 0.012 6 VNSSNSIKQ 0.000 2 MKLTVNSSN 0.000 9 SNSIKQRKP 0.000 V6-HLA-A3- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 FIVPKLEHI 0.203 1 EEIEFIVPK 0.182 9 KLEHIEQDE 0.090 2 EIEFIVPKL 0.081 6 IVPKLEHIE 0.002 7 VPKLEHIEQ 0.000 4 EFIVPKLEH 0.000 3 IEFIVPKLE 0.000 8 PKLEHIEQD 0.000 V7-HLA-A3- 9 mers 282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 19 HPEPPRWTK 1.350 16 FTLHPEPPR 0.450 17 TLHPEPPRW 0.300 10 NISHELFTL 0.270 5 VIVEDNISH 0.090 14 ELFTLHPEP 0.030 20 PEPPRWTKK 0.009 4 HVIVEDNIS 0.006 11 ISHELFTLH 0.005 6 IVEDNISHE 0.003 7 VEDNISHEL 0.003 3 FHVIVEDNI 0.001 8 EDNISHELF 0.001 1 HDFHVIVED 0.000 9 DNISHELFT 0.000 13 HELFTLHPE 0.000 12 SHELFTLHP 0.000 2 DFHVIVEDN 0.000 18 LHPEPPRWT 0.000 15 LFTLHPEPP 0.000

TABLE XIII V1-HLA-A3- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 14 LMFLLLKFSK 300.000 1111 LLLLTVCFVK 135.00 11 IVYLMFLLLK 90.000 187 DLYFANVEEK 90.000 546 HMLELHCESK 45.000 930 GVPEQPTFLK 40.500 870 SLLDGRTHPK 30.000 743 EMIIKWEPLK 27.000 4 LLLGRGLIVY 27.000 995 HLSNLNATTK 20.000 905 HLTVLAYNSK 20.000 532 KLRVSPKNPR 18.000 1112 LLLTVCFVKR 18.000 689 ILPLAPFVRY 18.000 342 IVEEPPRWTK 13.500 1037 KISGVNLTQK 13.500 9 GLIVYLMFLL 12.150 788 VMTPAVYAPY 9.000 1189 GLFSEDGSFI 9.000 312 KIENVSYQDK 6.000 30 SVQQVPTIIK 6.000 406 NLQPNHTAVY 6.000 126 IVPSVPKLPK 6.000 998 NLNATTKYFK 6.000 1073 SIFQDVIETR 4.500 274 LLLECFAEGL 4.050 158 GLPPLHIYWM 4.050 633 NLHLSERQNR 4.000 181 YMSQKGDLYF 4.000 785 TLRVMTPAVY 4.000 33 QVPTIIKQSK 3.000 219 PMKLVNSLK 3.000 62 NPEPTFSWTK 2.700 132 KLPKEKIDPL 2.700 688 VILPLAPFVR 2.700 212 RTIVQKMPMK 2.250 948 ATLSWGLPKK 2.250 509 SCWVENAIGH 2.000 733 NIRVQASQPK 2.000 1102 LMCAIALLTL 1.800 1001 ATTKYKFYLR 1.800 897 SLDAFSEFHL 1.800 114 KLGIAMSEEEI 1.800 1101 GLMCAIALLT 1.350 118 AMSEEIEFIV 1.350 691 PLAPFVRYQF 1.350 16 FLLLKFSKAI 1.350 283 LPTPQVDWNK 1.350 18 LLKFSKAIEI 1.200 170 ELEHIEQDER 1.200 848 TVPKDRVHGR 1.200 116 GIAMSEEIEF 1.200 947 TATLSWGLPK 1.200 105 GKYRCFASNK 0.900 967 LQYQIINDTY 0.900 5 LLGRGLIVYL 0.900 488 HIYENGTLQI 0.900 466 SPEAVVSWQK 0.900 598 TLEDQGIYCC 0.900 261 GSESSITILK 0.900 1107 ALLTLLLLTV 0.900 292 KIGGDLPKGR 0.900 1110 TLLLLTVCFV 0.900 209 KTLKIENVSY 0.900 957 KLNGNLTGYL 0.810 43 VQVAFPFDEY 0.810 471 VSWQKVEEVK 0.750 844 VTWSTVPKDR 0.750 481 PLEGRRYHIY 0.600 524 NLDIRNATKL 0.600 44 QVAFPFDEYF 0.600 529 NATKLRVSPK 0.600 701 RVIAVNEVGR 0.600 238 EIGSKANSIK 0.600 810 GPDPQSVTLY 0.540 10 LIVYLMFLLL 0.540 953 GLPKKLNGNL 0.540 738 ASQPKEMIIK 0.450 857 RLKGYQINWW 0.450 221 KLTVNSLKHA 0.450 123 IEFIVPSVPK 0.450 1088 GLYDDISTQG 0.450 150 VLPCNPPKGL 0.450 1136 HPDPEIQSVK 0.450 440 LIQTKDGENY 0.400 815 SVTLYSGEDY 0.400 559 HLKHSLKLSW 0.400 902 SEFHLTVLAY 0.360 1143 SVKDETFGEY 0.360 686 TTVILPLAPF 0.338 960 GNLTGYLLQY 0.324 148 PIVLPCNPPK 0.300 1108 LLTLLLLTVC 0.300 356 AVYSTGSNGI 0.300 426 ILANANIDVV 0.300 817 TLYSGEDYPD 0.300 535 VSPKNPRIPK 0.300 69 WFKDGNPFYF 0.300 840 TLVKVTWSTV 0.300 603 GIYCCSAHTA 0.300 V2-HLA-A3- 10 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 3 IVPSVPKFPK 9.000 6 SVPKFPKEKI 0.090 5 PSVPKFPKEK 0.034 2 FIVPSVPKFP 0.003 9 KFPKEKIDPL 0.003 1 EFIVPSVPKF 0.003 4 VPSVPKFPKE 0.001 10 FPKEKIDPLE 0.000 7 VPKFPKEKID 0.000 8 PKFPKEKIDP 0.000 V2-HLA-A3- 10 mers-(SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 TLGEGKYAGL 0.900 8 GKYAGLYDDI 0.009 3 STLGEGKYAG 0.007 5 LGEGKYAGLY 0.005 1 ESSTLGEGKY 0.002 2 SSTLGEGKYA 0.001 6 GEGKYAGLYD 0.000 7 EGKYAGLYDD 0.000 V2-HLA-A3- 10 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 TLGEGKYAGL 0.900 1 EESSTLGEGK 0.018 9 GKYAGLYDDI 0.009 4 STLGEGKYAG 0.007 6 LGEGKYAGLY 0.005 2 ESSTLGEGKY 0.002 10 KYAGLYDDIS 0.001 3 SSTLGEGKYA 0.001 7 GEGKYAGLYD 0.000 8 EGKYAGLYDD 0.000 V3-HLA-A3- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 46 NMLAEDFIQK 180.000 61 YVEKSSTFFK 6.000 52 FIQKSTSCNY 0.400 55 KSTSCNYVEK 0.300 47 MLAEDFIQKS 0.270 27 PQPSIFICSK 0.270 10 NTTYVSNTTY 0.200 39 ELSYRNRNML 0.180 23 ATGSPQPSIF 0.100 8 VINTTYVSNT 0.090 2 VIHGVDVINT 0.090 33 ICSKEQELSY 0.080 5 GVDVINTTYV 0.060 11 TTYVSNTTYV 0.050 34 CSKEQELSYR 0.045 59 CNYVEKSSTF 0.020 56 STSCNYVEKS 0.018 62 VEKSSTFFKI 0.016 25 GSPQPSIFIC 0.013 22 NATGSPQPSI 0.013 30 SIFCSKEQE 0.010 17 TTYVSNATGS 0.010 40 LSYRNRNMLA 0.010 4 HGVDVINTTY 0.009 14 VNSTTYVSNA 0.009 53 IQKSTSCNYV 0.006 26 SPQPSIFICS 0.005 36 KEQELSYRNR 0.005 60 NYVEKSSTFF 0.005 32 FICSKEQELS 0.004 43 RNRNMLAEDF 0.004 24 TGSPQPSIFI 0.003 19 YVSNATGSPQ 0.002 13 YVSNTTYVSN 0.002 16 NTTYVSNATG 0.001 58 SCNYVEKSST 0.001 31 IFICSKEQEL 0.001 7 DVINTTYVSN 0.001 48 LAEDFIQKST 0.001 44 NRNMLAEDFI 0.001 37 EQELSYRNRN 0.001 1 PVIHGVDVIN 0.000 28 QPSIFICSKE 0.000 15 SNTTYVSNAT 0.000 9 INTTYVSNTT 0.000 50 EDFIQKSTSC 0.000 3 IHGVDVINTT 0.000 45 RNMLAEDFIQ 0.000 12 TYVSNTTYVS 0.000 6 VDVINTTYVS 0.000 57 TSCNYVEKSS 0.000 49 AEDFIQKSTS 0.000 20 VSNATGSPQP 0.000 38 QELSYRNRNM 0.000 21 SNATGSPQPS 0.000 35 SKEQELSYRN 0.000 54 QKSTSCNYVE 0.000 41 SYRNRNMLAE 0.000 42 YRNRNMLAED 0.000 18 TYVSNATGSP 0.000 51 DFIQKSTSCN 0.000 29 PSIFICSKEQ 0.000 V4-HLA-A3- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 9 DLPEQPTFLK 40.500 3 TLYSGEDLPE 0.200 1 SVTLYSGEDL 0.060 7 GEDLPEQPTF 0.018 10 LPEQPTFLKV 0.012 2 VTLYSGEDLP 0.002 8 EDLPEQPTFL 0.000 6 SGEDLPEQPT 0.000 5 YSGEDLPEQP 0.000 4 LYSGEDLPEQ 0.000 V5-HLA-A3- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 KLTVNSSNSI 1.800 5 LTVNSSNSIK 1.500 8 NSSNSIKQRK 0.150 10 SNSIKQRKPK 0.020 7 VNSSNSIKQR 0.006 6 TVNSSNSIKQ 0.004 2 PMKLTVNSSN 0.003 1 MPMKLTVNSS 0.002 3 MKLTVNSSNS 0.000 9 SSNSIKQRKP 0.000 V6-HLA-A3- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 KLEHIEQDER 12.000 1 SEEIEFIVPK 0.270 4 IEFIVPKLEH 0.009 2 EEIEFIVPKL 0.005 6 FIVPKLEHIE 0.005 7 IVPKLEHIEQ 0.004 3 EIEFIVPKLE 0.000 5 EFIVPKLEHI 0.000 8 VPKLEHIEQD 0.000 9 PKLEHIEQDE 0.000

TABLE XIV V1-HLA- A1101-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 843 KVTWSTVPK 6.000 792 AVYAPYDVK 4.000 149 IVLPCNPPK 3.000 948 ATLSWGLPK 3.000 1118 FVKRNRGGK 2.000 835 DVINSTLVK 1.800 1112 LLLTVCFVK 1.800 906 LTVLAYNSK 1.500 739 SQPKEMIIK 1.200 760 GLEYRVTWK 1.200 12 VYLMFLLLK 1.200 106 KYRCFASNK 1.200 31 VQQVPTIIK 1.200 129 SVPKLPKEK 1.000 530 ATKLRVSPK 1.000 15 MFLLLKFSK 0.900 188 LYFANVEEK 0.800 1199 GAYAGSKEK 0.600 436 DVRPLIQTK 0.600 340 HVIVEEPPR 0.600 744 MIIKWEPLK 0.600 931 VPEQPTFLK 0.600 252 KANSIKQRK 0.600 661 YIVEFEGNK 0.600 867 KTKSLLDGR 0.600 213 TIVQKMPMK 0.600 861 YQINWWKTK 0.450 127 VPSVPKLPK 0.400 536 SPKNPRIPK 0.400 871 LLDGRTHPK 0.400 937 FLKVIKVDK 0.400 547 MLELHCESK 0.400 524 NLDIRNATK 0.400 520 AVTANLDIR 0.400 1002 TTKYKFYLR 0.400 949 TLSWGLPKK 0.400 1197 FIGAYAGSK 0.400 1150 GEYSDSDEK 0.360 301 RETKENYGK 0.360 1029 GEGSKGIGK 0.360 859 KGYQINWWK 0.240 689 ILPLAPFVR 0.240 34 VPTIIKQSK 0.200 52 YFQIECEAK 0.200 934 QPTFLKVIK 0.200 101 ACTSQGCGK 0.200 1 998 NLNATTKYK 0.200 284 PTPQVDWNK 0.200 304 KENYGKTLK 0.180 787 RVMTPAVYA 0.120 983 DINITTPSK 0.120 262 SESSITILK 0.120 478 EVKPLEGRR 0.120 392 FAGDVVFPR 0.120 343 VEEPPRWTK 0.120 296 DLPKGRETK 0.120 882 NILRFSGQR 0.120 202 YCCFAAFPR 0.120 677 ELTRVQGKK 0.120 98 GHISHFQGK 0.090 333 GTATHDFHV 0.090 212 RTIVQKMPM 0.090 779 ETVTNHTLR 0.090 124 EFIVPSVPK 0.090 702 VIAVNEVGR 0.080 74 NPFYFTDHR 0.080 11 IVYLMFLLL 0.080 877 HPKEVNILR 0.080 1113 LLTVCFVKR 0.080 396 VVFPREISF 0.080 693 APFVRYQFR 0.080 317 SYQDKGNYR 0.080 562 MSLKLSWSK 0.060 291 NKIGGDLPK 0.060 510 CWVENAIGK 0.060 680 RVQGKKTTV 0.060 764 RVTWKPQGA 0.060 942 KVDKDTATL 0.060 721 ETPPAAPDR 0.060 534 RVSPKNPRI 0.060 930 GVPEQPTFL 0.060 833 GVDVINSTL 0.060 313 IENVSYQDK 0.060 452 VVGYSAFLH 0.060 579 GTEDGRIII 0.060 424 GTILANANI 0.045 1115 TVCFVKRNR 0.040 780 TVTNHTLRV 0.040 214 IVQKMPMKL 0.040 849 VPKDRVHGR 0.040 1074 IFQDVIETR 0.040 9 GLIVYLMFL 0.036 247 KQRKPKLLL 0.036 734 IRVQASQPK 0.030 220 MKLTVNSLK 0.030 1124 GGKYSVKEK 0.030 685 KTTVILPLA 0.030 687 TVILPLAPF 0.030 1001 ATTKVKFVL 0.030 875 RTHPKEVNI 0.030 V2-HLA- A1101-9 mers-(SET 1)- Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 SVPKFPKEK 1.000 3 VPSVPKFPK 0.600 1 FIVPSVPKF 0.006 6 VPKFPKEKI 0.002 9 FPKEKIDPL 0.002 8 KFPKEKIDP 0.001 2 IVPSVPKFP 0.001 4 PSVPKFPKE 0.000 7 PKFPKEKID 0.000 V2-HLA- A1101-9 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 7 RAEKENYGK 0.360 2 DLPKGREAK 0.120 5 KGREAKENY 0.001 3 LPKGREAKE 0.000 9 AKENYGKTL 0.000 1 GDLPKGREA 0.000 6 GREAKENYG 0.000 8 EAKENYGKT 0.000 4 PKGREAKEN 0.000 V2-HLA- A1101-9 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 KYAGLYDDI 0.012 3 STLGEGKYA 0.007 1 ESSTLGEGK 0.006 6 GEGKYAGLY 0.002 4 TLGEGKYAG 0.001 8 GKYAGLYDD 0.000 5 LGEGKYAGL 0.000 2 SSTLGEGKY 0.000 7 EGKYAGLYD 0.000 V3-A1101- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 55 STSCNYVEK 1.000 46 MLAEDFIQK 0.800 27 QPSIFICSK 0.200 61 VEKSSTFFK 0.180 4 GVDVINTTY 0.060 60 YVEKSSTFF 0.020 10 TTYVSNTTY 0.020 22 ATGSPQPSI 0.010 40 SYRNRNMLA 0.008 52 IQKSTSCNY 0.006 59 NYVEKSSTF 0.006 11 TYVSNTTYV 0.006 12 YVSNTTYVS 0.004 31 FICSKEQEL 0.004 34 SKEQELSYR 0.004 36 EQELSYRNR 0.004 44 RNMLAEDFI 0.002 18 YVSNATGSP 0.002 16 TTYVSNATG 0.002 45 NMLAEDFIQ 0.002 6 DVINTTYVS 0.002 24 GSPQPSIFI 0.001 9 NTTYVSNTT 0.001 15 NTTYVSNAT 0.001 25 SPQPSIFIC 0.001 17 TYVSNATGS 0.001 33 CSKEQELSY 0.000 39 LSYRNRNML 0.000 51 FIQKSTSCN 0.000 14 SNTTYVSNA 0.000 7 VINTTYVSN 0.000 1 VIHGVDVIN 0.000 29 SIFICSKEQ 0.000 35 KEQELSYRN 0.000 30 IFICSKEQE 0.000 5 VDVINTTYV 0.000 47 LAEDFIQKS 0.000 43 NRNMLAEDF 0.000 32 ICSKEQELS 0.000 23 TGSPQPSIF 0.000 53 QKSTSCNYV 0.000 21 NATGSPQPS 0.000 62 EKSSTFFKI 0.000  26 PQPSIFICS 0.000  42 RNRNMLAED 0.000  54 KSTSCNYVE 0.000  38 ELSYRNRNM 0.000  57 SCNYVEKSS 0.000  37 QELSYRNRN 0.000  50 DFIQKSTSC 0.000 58 CNYVEKSST 0.000 20 SNATGSPQP 0.000 2 IHGVDVINT 0.000 8 INTTYVSNT 0.000 41 YRNRNMLAE 0.000 3 HGVDVINTT 0.000 48 AEDFIQKST 0.000 13 VSNTTYVSN 0.000 19 VSNATGSPQ 0.000 56 TSCNYVEKS 0.000 49 EDFIQKSTS 0.000 28 PSIFICSKE 0.000 V4-HLA- A1101-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 LPEQPTFLK 0.600 1 VTLYSGEDL 0.015 8 DLPEQPTFL 0.001 3 LYSGEDLPE 0.001 2 TLYSGEDLP 0.001 6 GEDLPEQPT 0.000 7 EDLPEQPTF 0.000 4 YSGEDLPEQ 0.000 5 SGEDLPEQP 0.000 V5-HLA- A1101-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 TVNSSNSIK 2.000 8 SSNSIKQRK 0.020 4 LTVNSSNIS 0.015 7 NSSNSIKQR 0.002 3 KLTVNSSNS 0.001 6 VNSSNSIKQ 0.000 1 PMKLTVNSS 0.000 2 MKLTVNSSN 0.000 9 SNSIKQRKP 0.000 V6-HLA- A1101-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 EEIEFIVPK 0.027 5 FIVPKLEHI 0.006 6 IVPKLEHIE 0.002 4 EFIVPKLEH 0.002 9 KLEHIEQDE 0.001 2 EIEFIVPKL 0.001 7 VPKLEHIEQ 0.000 3 IEFIVPKLE 0.000 8 PKLEHIEQD 0.000 V7-HLA- A1101-9 mers-282P1G3 Each peptide is a portion; of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 19 HPEPPRWTK 0.400 16 FTLHPEPPR 0.300 5 VIVEDNISH 0.012 10 NISHELFTL 0.012 20 PEPPRWTKK 0.006 17 TLHPEPPRW 0.004 4 HVIVEDNIS 0.003 6 IVEDNISHE 0.002 7 VEDNISHEL 0.001 3 FHVIVEDNI 0.000 14 ELFTLHPEP 0.000 11 ISHELFTLH 0.000 15 LFTLHPEPP 0.000 13 HELFTLHPE 0.000 2 DFHVIVEDN 0.000 8 EDNISHELF 0.000 1 HDFHVIVED 0.000 12 SHELFTLHP 0.000 9 DNISHELFT 0.000 18 LHPEPPRWT 0.000

TABLE XV V1-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 930 GVPEQPTFLK 18.000 11 IVYLMFLLLK 8.000 212 RTIVQKMPMK  4.500 342 IVEEPPRWTK  4.000 126 IVPSVPKLPK  4.000 30 SVQQVPTIIK  4.000 14 LMFLLLKFSK  2.400 33 QVPTIIKQSK  2.000 1111 LLLLTVCFVK  1.800 701 RVIAVNEVGR  1.800 948 ATLSWGLPKK  1.500 1037 KISGVNLTQK  1.200 312 KIENVSYQDK  1.200 509 SCWVENAIGK  0.800 1010 RACTSQGCGK  0.600 870 SLLDGRTHPK  0.600 860 GYQINWWKTK  0.600 546 HMLELHCESK  0.600 1001 ATTKYKFYLR 0.400 995 HLSNLNATTK  0.400 733 NIRVQASQPK  0.400 848 TVPKDRVHGR 0.400 947 TATLSWGLPK  0.400 283 LPTPQVDWNK  0.400 466 SPEAVVSWQK  0.400 905 HLTVLAYNSK 0.400 62 NPEPTFSWTK 0.400 688 VILPLAPFVR 0.360 1196 SFIGAYAGSK 0.300 936 TFLKVIKVDK 0.300 1117 CFVKRNRGGK 0.300 51 EYFQIECEAK 0.240 532 KLRVSPKNPR 0.240 187 DLYFANVEEK 0.240 1136 HPDPEIQSVK 0.200 208 FPRLRTIVQK 0.200 529 NATKLRVSPK 0.200 844 VTWSTVPKDR 0.200 933 EQPTFLKVIK 0.180 660 EYIVEFEGNK 0.180 743 EMIIKWEPLK 0.180 1073 SIFQDVIETR 0.160 1121 RNRGGKYSVK 0.120 105 GKYRCFASNK 0.120 561 KHSLKLSWSK 0.120 300 GRETKENYGK 0.120 261 GSESSITILK 0.120 123 IEFIVPSVPK 0.120 292 KIGGDLPKGR 0.120 238 EIGSKANSIK 0.120 1112 LLLTVCFVKR 0.120 1057 AEHIVRLMTK 0.120 179 RVYMSQKGDL 0.120 295 GDLPKGRETK 0.090 451 TVVGYSAFLH 0.090 939 KVIKVDKDTA 0.090 290 WNKIGGDLPK 0.080 633 NLHLSERQNR 0.080 201 DYCCFAAFPR 0.072 523 ANLDIRNATK 0.060 834 VDVINSTLVK 0.060 148 PIVLPCNPPK 0.060 519 TAVTANLDIR 0.060 680 RVQGKKTTVI 0.060 552 CESKCDSHLK 0.060 370 AEGEPQPTIK 0.060 381 RVNGSPVDNH 0.060 833 GVDVINSTLV 0.060 518 KTAVTANLDI 0.060 343 VEEPPRWTKK 0.060 90 GTFRIPNEGH 0.060 675 WEELTRVQGK 0.060 9 GLIVYLMFLL 0.054 309 KTLKIENVSY 0.045 692 LAPFVRYQFR 0.040 99 HISHFQGKYR 0.040 535 VSPKNPRIPK 0.040 858 LKGYQINWWK 0.040 738 ASQPKEMIIK 0.040 471 VSWQKVEEVK 0.040 356 AVYSTGSNGI 0.040 429 NANIDVVDVR 0.040 219 PMKLTVNSLK 0.040 84 IPSNNSGTFR 0.040 792 AVYAPYDVKV 0.040 1028 LGEGSKGIGK 0.040 190 FANVEEKDSR 0.040 726 APDRNPQNIR 0.040 413 AVYQCEASNV 0.040 639 RQNRSVRLTW 0.036 755 EQNGPGLEYR 0.036 842 VKVTWSTVPK 0.030 435 VDVRPLIQTK 0.030 982 NDINITTPSK 0.030 791 PAVYAPYDVK 0.030 997 SNLNATTKYK 0.030 1123 RGGKYSVKEK 0.030 340 HVIVEEPPRW 0.030 735 RVQASQPKEM 0.030 499 RTTEEDAGSY 0.030 V2-HLA- A1101-10 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 3 IVPSVPKFPK 6.000 6 SVPKFPKEKI 0.020 9 KFPKEKIDPL 0.006 5 PSVPKFPKEK 0.002 1 EFIVPSVPKF 0.001 2 FIVPSVPKFP 0.000 10 FPKEKIDPLE 0.000 4 VPSVPKFPKE 0.000 7 VPKFPKEKID 0.000 8 PKFPKEKIDP 0.000 V2-HLA- A1101-10 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 TLGEGKYAGL 0.004 3 STLGEGKYAG 0.003 8 GKYAGLYDDI 0.001 6 GEGKYAGLYD 0.000 5 LGEGKYAGLY 0.000 2 SSTLGEGKYA 0.000 1 ESSTLGEGKY 0.000 7 EGKYAGLYDD 0.000 V2-HLA- A1101-10 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 EESSTLGEGK 0.018 5 TLGEGKYAGL 0.004 4 STLGEGKYAG 0.003 9 GKYAGLYDDI 0.001 10 KYAGLYDDIS 0.001 7 GEGKYAGLYD 0.000 6 LGEGKYAGLY 0.000 3 SSTLGEGKYA 0.000 2 ESSTLGEGKY 0.000 8 EGKYAGLYDD 0.000 V3-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 61 YVEKSSTFFK 6.000 46 NMLAEDFIQK 1.200 55 KSTSCNYVEK 0.060 27 PQPSIFICSK 0.060 5 GVDVINTTYV 0.060 11 TTYVSNTTYV 0.020 10 NTTYVSNTTY 0.010 23 ATGSPQPSIF 0.010 60 NYVEKSSTFF 0.006 53 IQKSTSCNYV 0.006 33 ICSKEQELSY 0.004 34 CSKEQELSYR 0.004 52 FIQKSTSCNY 0.004 36 KEQELSYRNR 0.004 31 IFICSKEQEL 0.003 22 NATGSPQPSI 0.002 17 TTYVSNATGS 0.002 19 YVSNATGSPQ 0.002 13 YVSNTTYVSN 0.002 62 VEKSSTFFKI 0.002 12 TYVSNTTYVS 0.001 43 RNRNMLAEDF 0.001 39 ELSYRNRNML 0.001 56 STSCNYVEKS 0.001 16 NTTYVSNATG 0.001 7 DVINTTYVSN 0.001 30 SIFICSKEQE 0.001 41 SYRNRNMLAE 0.001 2 VIHGVDVINT 0.001 59 CNYVEKSSTF 0.001 40 LSYRNRNMLA 0.001 45 RNMLAEDFIQ 0.001 18 TYVSNATGSP 0.001 47 MLAEDFIQKS 0.000 24 TGSPQPSIFI 0.000 8 VINTTYVSNT 0.000 32 FICSKEQELS 0.000 26 SPQPSIFICS 0.000 4 HGVDVINTTY 0.000 1 PVIHGVDVIN 0.000 44 NRNMLAEDFI 0.000 28 QPSIFICSKE 0.000 58 SCNYVEKSST 0.000 14 VSNTTYVSNA 0.000 25 GSPQPSIFIC 0.000 37 EQELSYRNRN 0.000 48 LAEDFIQKST 0.000 51 DFIQKSTSCN 0.000 38 QELSYRNRNM 0.000 6 VDVINTTYVS 0.000 49 AEDFIQKSTS 0.000 54 QKSTSCNYVE 0.000 9 INTTYVSNTT 0.000 21 SNATGSPQPS 0.000 35 SKEQELSYRN 0.000 15 SNTTYVSNAT 0.000 20 VSNATGSPQP 0.000 3 IHGVDVINTT 0.000 42 YRNRNMLAED 0.000 50 EDFIQKSTSC 0.000 57 TSCNYVEKSS 0.000 29 PSIFICSKEQ 0.000 V4-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 9 DLPEQPTFLK 0.360 1 SVTLYSGEDL 0.020 10 LPEQPTFLKV 0.004 7 GEDLPEQPTF 0.002 3 TLYSGEDLPE 0.002 2 VTLYSGEDLP 0.002 4 LYSGEDLPEQ 0.000 8 EDLPEQPTFL 0.000 6 SGEDLPEQPT 0.000 5 YSGEDLPEQP 0.000 V5-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 LTVNSSNSIK 1.500 8 NSSNSIKQRK 0.020 10 SNSIKQRKPK 0.020 4 KLTVNSSNSI 0.012 6 TVNSSNSIKQ 0.004 7 VNSSNSIKQR 0.004 1 MPMKLTVNSS 0.000 2 PMKLTVNSSN 0.000 3 MKLTVNSSNS 0.000 9 SSNSIKQRKP 0.000 V6-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 KLEHIEQDER 0.240 1 SEEIEFIVPK 0.060 7 IVPKLEHIEQ 0.004 4 IEFIVPKLEH 0.002 5 EFIVPKLEHI 0.001 6 FIVPKLEHIE 0.001 2 EEIEFIVPKL 0.000 8 VPKLEHIEQD 0.000 3 EIEFIVPKLE 0.000 9 PKLEHIEQDE 0.000 V7-HLA- A1101-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 20 HPEPPRWTKK 0.200 5 HVIVEDNISH 0.060 16 LFTLHPEPPR 0.040 19 LHPEPPRWTK 0.040 7 IVEDNISHEL 0.020 17 FTLHPEPPRW 0.015 11 NISHELFTLH 0.004 6 VIVEDNISHE 0.001 8 VEDNISHELF 0.001 3 DFHVIVEDNI 0.001 10 DNISHELFTL 0.001 15 ELFTLHPEPP 0.000 14 HELFTLHPEP 0.000 18 TLHPEPPRWT 0.000 12 ISHELFTLHP 0.000 2 HDFHVIVEDN 0.000 4 FHVIVEDNIS 0.000 13 SHELFTLHPE 0.000 1 THDFHVIVED 0.000 9 EDNISHELFT 0.000 21 PEPPRWTKKP 0.000

TABLE XVI V1-HLA- A24-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 180 VYMSQKGDL 300.000 1182 EYGEGDHGL 240.000 323 NYRCTASNF 100.000 823 DYPDTAPVI 90.000 964 GYLLQYQII 90.000 489 IYENGTLQI 75.000 1085 EYAGLYDDI 60.000 357 VYSTGSNGI 60.000 76 FYFTDHRII 50.000 102 HFQGKYRCF 15.000 697 RYQFRVIAV 15.000 584 RIIIDGANL 12.000 486 RYHIYENGT 12.000 968 QYQIINDTY 10.500 1004 KYKFYLRAC 10.000 1052 VFEPGAEHI 9.000 1098 WFIGLMCAI 9.000 660 EYIVEFEGN 9.000 8 RGLIVYLMF 8.400 289 DWNKIGGDL 8.400 860 GYQINWWKT 8.250 991 KPSWHLSNL 8.000 942 KVDKDTATL 8.000 247 KQRKPKLLL 8.000 890 RNSGMVPSL 8.000 125 FIVPSVPKL 7.920 51 EYFQIECEA 7.700 414 VYQCEASNV 7.500 10 LIVYLMFLL 7.200 2 EPLLLGRGL 7.200 1094 STQGWFIGL 7.200 1104 CAIALLTLL 7.200 626 DVPDPPENL 7.200 930 GVPEQPTFL 7.200 448 NYATVVGYS 7.000 793 VYAPYDVKV 6.600 214 IVQKMPMKL 6.600 1100 IGLMCAIAL 6.000 451 TVVGYSAFL 6.000 1101 GLMCAIALL 6.000 1190 LFSEDGSFI 6.000 9 GLIVYLMFL 6.000 154 NPPKGLPPL 6.000 796 PYDVKVQAI 6.000 419 ASNVHGTIL 6.000 959 NGNLTGYLL 6.000 275 LLECFAEGL 6.000 261 GSESSITIL 6.000 267 TILKGEILL 6.000 753 SMEQNGPGL 6.000 901 FSEFHLTVL 6.000 743 EMIIKWEPL 6.000 266 ITILKGEIL 6.000 1127 YSVKEKEDL 6.000 1106 IALLTLLLL 6.000 833 GVDVINSTL 5.600 39 KQSKVQVAF 5.600 950 LSWGLPKKL 5.280 507 SYSCWVENA 5.000 109 CFASNKLGI 5.000 604 IYCCSAHTA 5.000 1172 QPTESADSL 4.800 946 DTATLSWGL 4.800 958 LNGNLTGYL 4.800 133 LPKEKIDPL 4.800 11 IVYLMFLLL 4.800 203 CCFAAFPRL 4.800 6 LGRGLIVYL 4.800 810 GPDPQSVTL 4.800 1105 AIALLTLLL 4.800 954 LPKKLNGNL 4.800 245 SIKQRKPKL 4.400 163 HIYWMNIEL 4.400 542 IPKLHMLEL 4.400 692 LAPFVRYQF 4.200 359 STGSNGILL 4.000 358 YSTGSNGIL 4.000 1103 MCAIALLTL 4.000 1152 YSDSDEKPL 4.000 1101 ATTKYKFYL 4.000 864 NWWKTKSLL 4.000 151 LPCNPPKGL 4.000 1035 IGKISGVNL 4.000 591 NLTISNVTL 4.000 682 QGKKTTVIL 4.000 1214 GSSTATFPL 4.000 863 INWWKTKSL 4.000 268 ILKGEILLL 4.000 605 YCCSAHTAL 4.000 988 TPSKPSWHL 4.000 13 YLMFLLLKF 3.960 893 GMVPSLDAF 3.600 1110 TLLLLTVCF 3.600 929 EGVPEQPTF 3.600 117 IAMSEEIEF 3.300 384 GSPVDNHPF 3.000 1183 YGEGDHGLF 3.000 450 ATVVGYSAF 3.000 687 TVILPLAPF 3.000 917 GPESEPYIF 3.000 V2-HLA- A24-9 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 FPKEKIDPL 4.800 1 FIVPSVPKF 3.960 6 VPKFPKEKI 1.100 8 KFPKEKIDP 0.150 2 IVPSVPKFP 0.021 5 SVPKFPKEK 0.017 3 VPSVPKFPK 0.010 4 PSVPKFPKE 0.002 7 PKFPKEKID 0.000 V2-HLA- A24-9 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 AKENYGKTL 0.600 5 KGREAKENY 0.240 8 EAKENYGKT 0.132 1 GDLPKGREA 0.020 2 DLPKGREAK 0.015 3 LPKGREAKE 0.011 7 REAKENYGK 0.002 6 GREAKENYG 0.002 4 PKGREAKEN 0.001 V2-HLA-A24- 9 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 KYAGLYDDI 120.000 5 LGEGKYAGL 6.000 3 STLGEGKYA 0.150 2 SSTLGEGKY 0.110 1 ESSTLGEGK 0.012 4 TLGEGKYAG 0.012 6 GEGKYAGLY 0.010 7 EGKYAGLYD 0.010 8 GKYAGLYDD 0.001 V3-HLA-A24- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 59 NYVEKSSTF 180.000 17 TYVSNATGS 7.500 11 TYVSNTTYV 7.500 31 FICSKEQEL 5.280 40 SYRNRNMLA 5.000 39 LSYRNRNML 4.800 60 YVEKSSTFF 3.000 44 RNMLAEDFI 3.000 23 TGSPQPSIF 2.400 24 GSPQPSIFI 1.500 22 ATGSPQPSI 1.000 50 DFIQKSTSC 0.750 38 ELSYRNRNM 0.500 43 NRNMLAEDF 0.360 3 HGVDVINTT 0.302 47 LAEDFIQKS 0.238 57 SCNYVEKSS 0.210 25 SPQPSIFIC 0.180 15 NTTYVSNAT 0.168 9 NTTYVSNTT 0.168 51 FIQKSTSCN 0.150 13 VSNTTYVSN 0.150 6 DVINTTYVS 0.150 7 VINTTYVSN 0.150 1 VIHGVDVIN 0.140 4 GVDVINTTY 0.140 62 EKSSTFFKI 0.132 33 CSKEQELSY 0.120 21 NATGSPQPS 0.120 56 TSCNYVEKS 0.110 10 TTYVSNTTY 0.100 14 SNTTYVSNA 0.100 32 ICSKEQELS 0.100 8 INTTYVSNT 0.100 12 YVSNTTYVS 0.100 52 IQKSTSCNY 0.100 58 CNYVEKSST 0.100 30 IFICSKEQE 0.075 35 KEQELSYRN 0.043 26 PQPSIFICS 0.025 42 RNRNMLAED 0.022 54 KSTSCNYVE 0.020 37 QELSYRNRN 0.018 19 VSNATGSPQ 0.015 36 EQELSYRNR 0.015 45 NMLAEDFIQ 0.015 5 VDVINTTYV 0.015 46 MLAEDFIQK 0.014 48 AEDFIQKST 0.014 53 QKSTSCNYV 0.012 55 STSCNYVEK 0.011 29 SIFICSKEQ 0.011 2 IHGVDVINT 0.010 16 TTYVSNATG 0.010 20 SNATGSPQP 0.010 27 QPSIFICSK 0.010 18 YVSNATGSP 0.010 49 EDFIQKSTS 0.010 28 PSIFICSKE 0.002 34 SKEQELSYR 0.002 41 YRNRNMLAE 0.002 61 VEKSSTFFK 0.001 V4-HLA-A24- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 8 DLPEQPTFL 7.200 1 VTLYSGEDL 6.000 3 LYSGEDLPE 0.500 7 EDLPEQPTF 0.360 5 SGEDLPEQP 0.022 9 LPEQPTFLK 0.015 4 YSGEDLPEQ 0.013 6 GEDLPEQPT 0.012 2 TLYSGEDLP 0.010 V5-HLA-A24- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 4 LTVNSSNSI 1.800 3 KLTVNSSNS 0.200 8 SSNSIKQRK 0.025 2 MKLTVNSSN 0.021 5 TVNSSNSIK 0.015 1 PMKLTVNSS 0.012 6 VNSSNSIKQ 0.011 9 SNSIKQRKP 0.011 7 NSSNSIKQR 0.010 V6-HLA-A24- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 EIEFIVPKL 9.240 5 FIVPKLEHI 1.800 4 EFIVPKLEH 0.083 9 KLEHIEQDE 0.050 6 IVPKLEHIE 0.018 7 VPKLEHIEQ 0.011 1 EEIEFIVPK 0.002 3 IEFIVPKLE 0.001 8 PKLEHIEQD 0.000 V7-HLA-A24- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 10 NISHELFTL 4.000 2 DFHVIVEDN 0.700 7 VEDNISHEL 0.616 8 EDNISHELF 0.300 3 FHVIVEDNI 0.210 4 HVIVEDNIS 0.180 9 DNISHELFT 0.150 17 TLHPEPPRW 0.120 15 LFTLHPEPP 0.050 18 LHPEPPRWT 0.018 6 IVEDNISHE 0.018 5 VIVEDNISH 0.018 19 HPEPPRWTK 0.018 11 ISHELFTLH 0.017 16 FTLHPEPPR 0.015 14 ELFTLHPEP 0.013 1 HDFHVIVED 0.002 13 HELFTLHPE 0.002 12 SHELFTLHP 0.002 20 PEPPRWTKK 0.000

TABLE XVII V1-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 106 KYRCFASNKL 528.000 1126 KYSVKEKEDL 400.000 486 RYHIYENGTL 400.000 323 NYRCTASNFL 240.000 1151 EYSDSDEKPL 240.000 357 VYSTGSNGIL 200.000 604 IYCCSAHTAL 200.000 12 VYLMFLLLKF 198.000 454 GYSAFLHCEF 132.000 1182 EYGEGDHGLF 120.000 975 TYEIGELNDI 90.000 507 SYSCWVENAI 84.000 124 EFIVPSVPKL 33.000 900 AFSEFHLTVL 24.000 697 RYQFRVIAVN 21.000 330 NFLGTATHDF 15.000 752 KSMEQNGPGL 14.400 957 KLNGNLTGYL 14.400 541 RIPKLHMLEL 13.200 1019 KPITEESSTL 12.000 1158 KPLKGSLRSL 12.000 132 KLPKEKIDPL 12.000 8 RGLIVYLMFL 12.000 1004 KYKFYLRACT 12.000 875 RTHPKEVNIL 11.520 832 HGVDVINSTL 10.080 555 KCDSHLKHSL 9.600 489 IYENGTLQIN 9.000 317 SYQDKGNYRC 9.000 179 RVYMSQKGDL 8.000 180 VYMSQKGDLY 7.500 964 GYLLQYQIIN 7.500 46 AFPFDEYFQI 7.500 1089 LYDDISTQGW 7.200 153 CNPPKGLPPL 7.200 9 GLIVYLMFLL 7.200 929 EGVPEQPTFL 7.200 218 MPMKLTVNSL 7.200 953 GLPKKLNGNL 7.200 274 LLLECFAEGL 7.200 142 EVEEGDPIVL 7.200 10 LIVYLMFLLL 7.200 809 SGPDPQSVTL 7.200 270 KGEILLLECF 7.200 1104 CAIALLTLLL 7.200 448 NYATVVGYSA 7.000 849 VPKDRVHGRL 6.720 213 TIVQKMPMKL 6.600 306 NYGKTLKIEN 6.600 244 NSIKQRKPKL 6.600 1100 IGLMCAIALL 6.000 551 HCESKCDSHL 6.000 590 ANLTISNVTL 6.000 266 ITILKGEILL 6.000 267 TILKGEILLL 6.000 150 VLPCNPPKGL 6.000 987 TTPSKPSWHL 6.000 862 QINWWKTKSL 6.000 1171 MQPTESADSL 6.000 450 ATVVGYSAFL 6.000 398 FPREISFTNL 5.760 516 IGKTAVTANL 5.600 949 TLSWGLPKKL 5.280 1200 AYAGSKEKGS 5.000 818 LYSGEDYPDT 5.000 885 RFSGQRNSGM 5.000 91 TFRIPNEGHI 5.000 1085 EYAGLYDDIS 5.000 260 SGSESSITIL 4.800 798 DVKVQAINQL 4.800 627 VPDPPENLHL 4.800 1093 ISTQGWFIGL 4.800 302 ETKENYGKTL 4.800 202 YCCFAAFPRL 4.800 5 LLGRGLIVYL 4.800 1103 MCAIALLTLL 4.800 199 RNDYCCFAAF 4.800 536 SPKNPRIPKL 4.400 557 DSHLKHSLKL 4.400 972 INDTYEIGEL 4.400 524 NLDIRNATKL 4.400 681 VQGKKTTVIL 4.000 245 SIKQRKPKLL 4.000 539 NPRIPKLHML 4.000 1034 GIGKISGVNL 4.000 1105 AIALLTLLLL 4.000 431 NIDVVDVRPL 4.000 863 INWWKTKSLL 4.000 1099 FIGLMCAIAL 4.000 1102 LMCAIALLTL 4.000 1213 NGSSTATFPL 4.000 1054 EPGAEHIVRL 4.000 958 LNGNLTGYLL 4.000 897 SLDAFSEFHL 4.000 1066 KNWGDNDSIF 4.000 418 EASNVHGTIL 4.000 1000 NATTKYKFYL 4.000 358 YSTGSNGILL 4.000 1080 ETRGREYAGL 4.000 616 AADITQVTVL 4.000 V2-HLA- A24-10 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 9 KFPKEKIDPL 60.000 1 EFIVPSVPKF 16.500 6 SVPKFPKEKI 1.650 2 FIVPSVPKFP 0.025 10 FPKEKIDPLE 0.017 3 IVPSVPKFPK 0.015 4 VPSVPKFPKE 0.013 7 VPKFPKEKID 0.010 5 PSVPKFPKEK 0.002 8 PKFPKEKIDP 0.000 V2-HLA- A24-10 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 4 TLGEGKYAGL 4.800 5 LGEGKYAGLY 0.150 8 GKYAGLYDDI 0.120 1 ESSTLGEGKY 0.110 2 SSTLGEGKYA 0.100 3 STLGEGKYAG 0.015 7 EGKYAGLYDD 0.010 6 GEGKYAGLYD 0.001 V2-HLA- A24-10 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 10 KYAGLYDDIS 10.000 5 TLGEGKYAGL 4.800 6 LGEGKYAGLY 0.150 9 GKYAGLYDDI 0.120 2 ESSTLGEGKY 0.110 3 SSTLGEGKYA 0.100 4 STLGEGKYAG 0.015 8 EGKYAGLYDD 0.010 1 EESSTLGEGK 0.001 7 GEGKYAGLYD 0.001 V3-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 60 NYVEKSSTFF 180.000 31 IFICSKEQEL 39.600 12 TYVSNTTYVS 7.500 43 RNRNMLAEDF 4.800 39 ELSYRNRNML 4.800 23 ATGSPQPSIF 2.000 59 CNYVEKSSTF 2.000 24 TGSPQPSIFI 1.200 22 NATGSPQPSI 1.000 51 DFIQKSTSCN 0.750 18 TYVSNATGSP 0.750 41 SYRNRNMLAE 0.500 26 SPQPSIFICS 0.302 4 HGVDVINTTY 0.252 48 LAEDFIQKST 0.252 37 EQELSYRNRN 0.180 15 SNTTYVSNAT 0.168 9 INTTYVSNTT 0.168 47 MLAEDFIQKS 0.158 25 GSPQPSIFIC 0.150 8 VINTTYVSNT 0.150 14 VSNTTYVSNA 0.150 7 DVINTTYVSN 0.150 44 NRNMLAEDFI 0.150 52 FIQKSTSCNY 0.150 58 SCNYVEKSST 0.150 57 TSCNYVEKSS 0.140 62 VEKSSTFFKI 0.132 53 IQKSTSCNYV 0.120 21 SNATGSPQPS 0.120 56 STSCNYVEKS 0.110 17 TTYVSNATGS 0.100 10 NTTYVSNTTY 0.100 2 VIHGVDVINT 0.100 5 GVDVINTTYV 0.100 11 TTYVSNTTYV 0.100 32 FICSKEQELS 0.100 33 ICSKEQELSY 0.100 13 YVSNTTYVSN 0.100 40 LSYRNRNMLA 0.100 38 QELSYRNRNM 0.075 45 RNMLAEDFIQ 0.030 55 KSTSCNYVEK 0.022 1 PVIHGVDVIN 0.021 35 SKEQELSYRN 0.018 46 NMLAEDFIQK 0.018 3 IHGVDVINTT 0.017 28 QPSIFICSKE 0.015 20 VSNATGSPQP 0.015 6 VDVINTTYVS 0.015 61 YVEKSSTFFK 0.015 34 CSKEQELSYR 0.012 50 EDFIQKSTSC 0.010 49 AEDFIQKSTS 0.010 16 NTTYVSNATG 0.010 19 YVSNATGSPQ 0.010 30 SIFICSKEQE 0.010 36 KEQELSYRNR 0.004 29 PSIFICSKEQ 0.002 42 YRNRNMLAED 0.002 27 PQPSIFICSK 0.002 54 QKSTSCNYVE 0.001 V4-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 1 SVTLYSGEDL 4.000 8 EDLPEQPTFL 0.720 4 LYSGEDLPEQ 0.550 6 SGEDLPEQPT 0.216 7 GEDLPEQPTF 0.200 10 LPEQPTFLKV 0.198 9 DLPEQPTFLK 0.018 2 VTLYSGEDLP 0.015 5 YSGEDLPEQP 0.014 3 TLYSGEDLPE 0.010 V5-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 4 KLTVNSSNSI 2.400 1 MPMKLTVNSS 0.180 8 NSSNSIKQRK 0.017 6 TVNSSNSIKQ 0.017 9 SSNSIKQRKP 0.017 3 MKLTVNSSNS 0.015 5 LTVNSSNSIK 0.015 2 PMKLTVNSSN 0.014 7 VNSSNSIKQR 0.010 10 SNSIKQRKPK 0.010 V6-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 5 EFIVPKLEHI 7.500 2 EEIEFIVPKL 1.109 10 KLEHIEQDER 0.033 6 FIVPKLEHIE 0.022 3 EIEFIVPKLE 0.021 7 IVPKLEHIEQ 0.017 8 VPKLEHIEQD 0.010 1 SEEIEFIVPK 0.002 4 IEFIVPKLEH 0.001 9 PKLEHIEQDE 0.000 V7-HLA- A24-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine Start Subsequence Score 7 IVEDNISHEL 11.088 3 DFHVIVEDNI 7.000 10 DNISHELFTL 6.000 8 VEDNISHELF 0.200 17 FTLHPEPPRW 0.150 18 TLHPEPPRWT 0.120 16 LFTLHPEPPR 0.050 20 HPEPPRWTKK 0.020 4 FHVIVEDNIS 0.018 6 VIVEDNISHE 0.018 5 HVIVEDNISH 0.015 9 EDNISHELFT 0.015 11 NISHELFTLH 0.014 2 HDFHVIVEDN 0.014 12 ISHELFTLHP 0.012 15 ELFTLHPEPP 0.010 14 HELFTLHPEP 0.002 19 LHPEPPRWTK 0.002 1 THDFHVIVED 0.002 13 SHELFTLHPE 0.002 21 PEPPRWTKKP 0.000

TABLE XVIII V1-HLA-B7- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 539 NPRIPKLHM 300.000 151 LPCNPPKGL 120.000 988 TPSKPSWHL 120.000 2 EPLLLGRGL 80.000 133 LPKEKIDPL 80.000 991 KPSWHLSNL 80.000 1172 QPTESADSL 80.000 542 IPKLHMLEL 80.000 954 LPKKLNGNL 80.000 154 NPPKGLPPL 80.000 247 KQRKPKLLL 60.000 6 LGRGLIVYL 40.000 626 DVPDPPENL 30.000 810 GPDPQSVTL 24.000 695 FVRYQFRVI 20.000 11 IVYLMFLLL 20.000 214 IVQKMPMKL 20.000 930 GVPEQPTFL 20.000 451 TVVGYSAFL 20.000 159 LPPLHIYWM 20.000 1106 IALLTLLLL 12.000 1101 GLMCAIALL 12.000 1001 ATTKYKFYL 12.000 130 VPKLPKEKI 12.000 828 APVIHGVDV 12.000 1105 AIALLTLLL 12.000 1104 CAIALLTLL 12.000 419 ASNVHGTIL 12.000 1163 SLRSLNRDM 10.000 210 RLRTIVQKM 10.000 285 TPQVDWNKI 8.000 47 FPFDEYFQI 8.000 726 APDRNPQNI 7.200 833 GVDVINSTL  6.000 772 APVEWEEET  6.000 942 KVDKDTATL  6.000 795 APYDVKVQA  6.000 1100 IGLMCAIAL 4.000 398 FPREISFTN 4.000 863 INWWKTKSL 4.000 267 TILKGEILL 4.000 9 GLIVYLMFL 4.000 946 DTATLSWGL 4.000 591 NLTISNVTL 4.000 10 LIVYLMFLL 4.000 268 ILKGEILLL 4.000 1127 YSVKEKEDL 4.000 950 LSWGLPKKL 4.000 266 ITILKGEIL 4.000 1214 GSSTATFPL 4.000 203 CCFAAFPRL 4.000 1103 MCAIALLTL 4.000 959 NGNLTGYLL 4.000 358 YSTGSNGIL 4.000 605 YCCSAHTAL 4.000 743 EMIIKWEPL 4.000 584 RIIIDGANL 4.000 125 FIVPSVPKL 4.000 163 HIYWMNIEL 4.000 890 RNSGMVPSL 4.000 1094 STQGWFIGL 4.000 359 STGSNGILL 4.000 1035 IGKISGVNL 4.000 682 QGKKTTVIL 4.000 245 SIKQRKPKL 4.000 958 LNGNLTGYL 4.000 855 HGRLKGYQI 4.000 206 AAFPRLRTI 3.600 730 NPQNIRVQA 3.000 111 ASNKLGIAM 3.000 433 DVVDVRPLI 3.000 787 RVMTPAVYA 2.250 250 KPKLLLPPT 2.000 758 GPGLEYRVT 2.000 352 KPQSAVYST 2.000 208 FPRLRTIVQ 2.000 596 NVTLEDQGI 2.000 534 RVSPKNPRI 2.000 30 SVQQVPTII 2.000 785 TLRVMTPAV 2.000 385 SPVDNHPFA 2.000 671 EPGRWEELT 2.000 873 DGRTHPKEV 2.000 1019 KPITEESST 2.000 1121 RNRGGKYSV 2.000 737 QASQPKEMI 1.800 753 SMEQNGPGL 1.200 334 TATHDFHVI 1.200 916 AGPESEPYI 1.200 118 AMSEEIEFI 1.200 519 TAVTANLDI 1.200 180 VYMSQKGDL 1.200 261 GSESSITIL 1.200 738 ASQPKEMII 1.200 650 AGADHNSNI 1.200 611 TALDSAADI 1.200 901 FSEFHLTVL 1.200 1152 YSDSDEKPL 1.200 218 MPMKLTVNS 1.200 418 EASNVHGTI 1.200 V2-HLA-B7- 9 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 FPKEKIDPL 80.000 6 VPKFPKEKI 12.000 3 VPSVPKFPK 0.300 5 SVPKFPKEK 0.050 2 IVPSVPKFP 0.050 1 FIVPSVPKF 0.020 4 PSVPKFPKE 0.001 8 KFPKEKIDP 0.001 7 PKFPKEKID 0.000 V2-HLA-B7- 9 mers-(SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 AKENYGKTL 0.360 8 EAKENYGKT 0.300 5 KGREAKENY 0.200 3 LPKGREAKE 0.200 2 DLPKGREAK 0.015 1 GDLPKGREA 0.010 7 REAKENYGK 0.001 6 GREAKENYG 0.000 4 PKGREAKEN 0.000 V2-HLA-B7- 9 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 LGEGKYAGL 1.200 3 STLGEGKYA 0.100 9 KYAGLYDDI 0.040 2 SSTLGEGKY 0.020 7 EGKYAGLYD 0.010 1 ESSTLGEGK 0.010 4 TLGEGKYAG 0.010 6 GEGKYAGLY 0.002 8 GKYAGLYDD 0.001 V3-HLA-B7- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 39 LSYRNRNML 6.000 31 FICSKEQEL 4.000 25 SPQPSIFIC 2.000 22 ATGSPQPSI 1.800 44 RNMLAEDFI  1.200 38 ELSYRNRNM 1.000 24 GSPQPSIFI 0.600 27 QPSIFICSK 0.200 8 INTTYVSNT 0.100 15 NTTYVSNAT 0.100 3 HGVDVINTT 0.100 12 YVSNTTYVS 0.100 58 CNYVEKSST 0.100 42 RNRNMLAED 0.100 6 DVINTTYVS 0.100 40 SYRNRNMLA 0.100 14 SNTTYVSNA 0.100 9 NTTYVSNTT 0.100 21 NATGSPQPS 0.060 18 YVSNATGSP 0.050 62 EKSSTFFKI 0.040 60 YVEKSSTFF 0.030 4 GVDVINTTY 0.030 10 TTYVSNTTY 0.020 32 ICSKEQELS 0.020 56 TSCNYVEKS 0.020 5 VDVINTTYV 0.020 13 VSNTTYVSN 0.020 51 FIQKSTSCN 0.020 53 QKSTSCNYV 0.020 33 CSKEQELSY 0.020 1 VIHGVDVIN 0.020 57 SCNYVEKSS 0.020 23 TGSPQPSIF 0.020 7 VINTTYVSN 0.020 52 IQKSTSCNY 0.020 11 TYVSNTTYV 0.020 47 LAEDFIQKS 0.018 16 TTYVSNATG 0.010 2 IHGVDVINT 0.010 46 MLAEDFIQK 0.010 45 NMLAEDFIQ 0.010 19 VSNATGSPQ 0.010 20 SNATGSPQP 0.010 54 KSTSCNYVE 0.010 50 DFIQKSTSC 0.010 29 SIFICSKEQ 0.010 55 STSCNYVEK 0.010 48 AEDFIQKST 0.009 37 QELSYRNRN 0.003 36 EQELSYRNR 0.003 59 NYVEKSSTF 0.002 26 PQPSIFICS 0.002 43 NRNMLAEDF 0.002 35 KEQELSYRN 0.002 17 TYVSNATGS 0.002 49 EDFIQKSTS 0.002 61 VEKSSTFFK 0.001 28 PSIFICSKE 0.001 41 YRNRNMLAE 0.001 30 IFICSKEQE 0.001 34 SKEQELSYR 0.000 V4-HLA-B7- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 VTLYSGEDL 4.000 8 DLPEQPTFL 4.000 9 LPEQPTFLK 0.090 4 YSGEDLPEQ 0.010 2 TLYSGEDLP 0.010 6 GEDLPEQPT 0.004 5 SGEDLPEQP 0.003 7 EDLPEQPTF 0.002 3 LYSGEDLPE 0.001 V4-HLA-B7- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 1 VTLYSGEDL 4.000 8 DLPEQPTFL 4.000 9 LPEQPTFLK 0.090 4 YSGEDLPEQ 0.010 2 TLYSGEDLP 0.010 6 GEDLPEQPT 0.004 5 SGEDLPEQP 0.003 7 EDLPEQPTF 0.002 3 LYSGEDLPE 0.001 V5-HLA- B7-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 4 LTVNSSNSI 0.400 5 TVNSSNSIK 0.050 3 KLTVNSSNS 0.020 8 SSNSIKQRK 0.010 6 VNSSNSIKQ 0.010 7 NSSNSIKQR 0.010 9 SNSIKQRKP 0.010 2 MKLTVNSSN 0.002 1 PMKLTVNSS 0.002 V6-HLA- B7-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 EIEFIVPKL 1.200 5 FIVPKLEHI 0.400 7 VPKLEHIEQ 0.200 6 IVPKLEHIE 0.050 9 KLEHIEQDE 0.003 4 EFIVPKLEH 0.002 3 IEFIVPKLE 0.001 1 EEIEFIVPK 0.001 8 PKLEHIEQD 0.000 V7-HLA- B7-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 10 NISHELFTL 4.000 19 HPEPPRWTK 0.135 7 VEDNISHEL 0.120 4 HVIVEDNIS 0.100 9 DNISHELFT 0.100 3 FHVIVEDNI 0.040 17 TLHPEPPRW 0.020 16 FTLHPEPPR 0.015 6 IVEDNISHE 0.015 18 LHPEPPRWT 0.015 11 ISHELFTLH 0.010 14 ELFTLHPEP 0.010 5 VIVEDNISH 0.010 8 EDNISHELF 0.002 2 DFHVIVEDN 0.002 13 HELFTLHPE 0.001 1 HDFHVIVED 0.001 15 LFTLHPEPP 0.001 12 SHELFTLHP 0.000 20 PEPPRWTKK 0.000

TABLE XIX V1-HLA-B7- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 398 FPREISFTNL 800.000 539 NPRIPKLHML 800.000 218 MPMKLTVNSL 240.000 1054 EPGAEHIVRL 80.000 1158 KPLKGSLRSL 80.000 849 VPKDRVHGRL 80.000 536 SPKNPRIPKL 80.000 1019 KPITEESSTL 80.000 1080 ETRGREYAGL 40.000 828 APVIHGVDVI 24.000 627 VPDPPENLHL 24.000 795 APYDVKVQAI 24.000 798 DVKVQAINQL 20.000 179 RVYMSQKGDL 20.000 1105 AIALLTLLLL 12.000 693 APFVRYQFRV 12.000 752 KSMEQNGPGL 12.000 450 ATVVGYSAFL 12.000 772 APVEWEEETV 12.000 1000 NATTKYKFYL 12.000 480 KPLEGRRYHI 12.000 590 ANLTISNVTL 12.000 418 EASNVHGTIL 12.000 2 EPLLLGRGLI 12.000 1104 CAIALLTLLL 12.000 616 AADITQVTVL 10.800 6 LGRGLIVYLM 10.000 74 NPFYFTDHRI 8.000 695 FVRYQFRVIA 7.500 356 AVYSTGSNGI 6.000 150 VLPCNPPKGL 6.000 142 EVEEGDPIVL 6.000 987 TTPSKPSWHL 6.000 643 SVRLTWEAGA 5.000 735 RVQASQPKEM 5.000 780 TVTNHTLRVM 5.000 863 INWWKTKSLL 4.000 790 TPAVYAPYDV 4.000 1034 GIGKISGVNL 4.000 266 ITILKGEILL 4.000 1103 MCAIALLTLL 4.000 9 GLIVYLMFLL 4.000 953 GLPKKLNGNL 4.000 323 NYRCTASNFL 4.000 106 KYRCFASNKL 4.000 862 QINWWKTKSL 4.000 274 LLLECFAEGL 4.000 541 RIPKLHMLEL 4.000 260 SGSESSITIL 4.000 949 TLSWGLPKKL 4.000 213 TIVQKMPMKL 4.000 557 DSHLKHSLKL 4.000 957 KLNGNLTGYL 4.000 1102 LMCAIALLTL 4.000 1093 ISTQGWFIGL 4.000 934 QPTFLKVIKV 4.000 132 KLPKEKIDPL 4.000 5 LLGRGLIVYL 4.000 8 RGLIVYLMFL 4.000 832 HGVDVINSTL 4.000 1099 FIGLMCAIAL 4.000 267 TILKGEILLL 4.000 929 EGVPEQPTFL 4.000 516 IGKTAVTANL 4.000 202 YCCFAAFPRL 4.000 473 WQKVEEVKPL 4.000 244 NSIKQRKPKL 4.000 373 EPQPTIKWRV 4.000 1171 MQPTESADSL 4.000 302 ETKENYGKTL 4.000 681 VQGKKTTVIL 4.000 1213 NGSSTATFPL 4.000 1100 IGLMCAIALL 4.000 809 SGPDPQSVTL 4.000 958 LNGNLTGYLL 4.000 358 YSTGSNGILL 4.000 637 SERQNRSVRL 4.000 153 CNPPKGLPPL 4.000 875 RTHPKEVNIL 4.000 1172 QPTESADSLV 4.000 265 SITILKGEIL 4.000 245 SIKQRKPKLL 4.000 34 VPTIIKQSKV 4.000 10 LIVYLMFLLL 4.000 725 AAPDRNPQNI 3.600 117 IAMSEEIEFI 3.600 110 FASNKLGIAM 3.000 792 AVYAPYDVKV 3.000 413 AVYQCEASNV 3.000 129 SVPKLPKEKI 3.000 206 AAFPRLRTIV 2.700 1048 HPIEVFEPGA 2.000 680 RVQGKKTTVI 2.000 27 IPSSVQQVPT 2.000 526 DIRNATKLRV 2.000 408 QPNHTAVYQC 2.000 1051 EVFEPGAEHI 2.000 954 LPKKLNGNLT 2.000 208 FPRLRTIVQK 2.000 538 KNPRIPKLHM 1.500 V2-HLA-B7- l0 mers-(SET 1)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 6 SVPKFPKEKI 3.000 9 KFPKEKIDPL 0.400 7 VPKFPKEKID 0.200 4 VPSVPKFPKE 0.200 10 FPKEKIDPLE 0.200 3 IVPSVPKFPK 0.075 2 FIVPSVPKFP 0.010 1 EFIVPSVPKF 0.002 5 PSVPKFPKEK 0.001 8 PKFPKEKIDP 0.000 V2-HLA-B7- l0 mers-(SET 2)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 4 TLGEGKYAGL 4.000 2 SSTLGEGKYA 0.100 8 GKYAGLYDDI 0.040 1 ESSTLGEGKY 0.020 3 STLGEGKYAG 0.010 7 EGKYAGLYDD 0.010 5 LGEGKYAGLY 0.006 6 GEGKYAGLYD 0.001 V2-HLA-B7- l0 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 5 TLGEGKYAGL 4.000 3 SSTLGEGKYA 0.100 9 GKYAGLYDDI 0.040 2 ESSTLGEGKY 0.020 4 STLGEGKYAG 0.010 8 EGKYAGLYDD 0.010 6 LGEGKYAGLY 0.006 10 KYAGLYDDIS 0.002 7 GEGKYAGLYD 0.001 1 EESSTLGEGK 0.001 V3-HLA-B7- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 39 ELSYRNRNML 6.000 22 NATGSPQPSI 1.800 24 TGSPQPSIFI 0.600 31 IFICSKEQEL 0.400 26 SPQPSIFICS 0.400 5 GVDVINTTYV 0.300 53 IQKSTSCNYV 0.200 28 QPSIFICSKE 0.200 11 TTYVSNTTYV 0.200 43 RNRNMLAEDF 0.200 8 VINTTYVSNT 0.100 15 SNTTYVSNAT 0.100 58 SCNYVEKSST 0.100 38 QELSYRNRNM 0.100 9 INTTYVSNTT 0.100 14 VSNTTYVSNA 0.100 7 DVINTTYVSN 0.100 2 VIHGVDVINT 0.100 25 GSPQPSIFIC 0.100 40 LSYRNRNMLA 0.100 13 YVSNTTYVSN 0.100 48 LAEDFIQKST 0.090 23 ATGSPQPSIF 0.060 19 YVSNATGSPQ 0.050 62 VEKSSTFFKI 0.040 44 NRNMLAEDFI 0.040 45 RNMLAEDFIQ 0.030 17 TTYVSNATGS 0.020 47 MLAEDFIQKS 0.020 10 NTTYVSNTTY 0.020 32 FICSKEQELS 0.020 59 CNYVEKSSTF 0.020 21 SNATGSPQPS 0.020 52 FIQKSTSCNY 0.020 56 STSCNYVEKS 0.020 4 HGVDVINTTY 0.020 57 TSCNYVEKSS 0.020 33 ICSKEQELSY 0.020 61 YVEKSSTFFK 0.015 16 NTTYVSNATG 0.010 1 PVIHGVDVIN 0.010 20 VSNATGSPQP 0.010 41 SYRNRNMLAE 0.010 34 CSKEQELSYR 0.010 30 SIFICSKEQE 0.010 50 EDFIQKSTSC 0.010 55 KSTSCNYVEK 0.010 3 IHGVDVINTT 0.010 46 NMLAEDFIQK 0.010 37 EQELSYRNRN 0.009 60 NYVEKSSTFF 0.002 51 DFIQKSTSCN 0.002 12 TYVSNTTYVS 0.002 6 VDVINTTYVS 0.002 49 AEDFIQKSTS 0.002 36 KEQELSYRNR 0.001 29 PSIFICSKEQ 0.001 54 QKSTSCNYVE 0.001 27 PQPSIFICSK 0.001 42 YRNRNMLAED 0.001 18 TYVSNATGSP 0.001 35 SKEQELSYRN 0.001 V4-HLA-B7- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 SVTLYSGEDL 20.000 10 LPEQPTFLKV 1.200 8 EDLPEQPTFL 0.400 6 SGEDLPEQPT 0.045 9 DLPEQPTFLK 0.015 3 TLYSGEDLPE 0.010 5 YSGEDLPEQP 0.010 2 VTLYSGEDLP 0.010 4 LYSGEDLPEQ 0.001 7 GEDLPEQPTF 0.001 V5-HLA- B7-l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position  is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 MPMKLTVNSS 1.200 4 KLTVNSSNSI 0.400 6 TVNSSNSIKQ 0.050 10 SNSIKQRKPK 0.015 7 VNSSNSIKQR 0.010 8 NSSNSIKQRK 0.010 9 SSNSIKQRKP 0.010 5 LTVNSSNSIK 0.010 2 PMKLTVNSSN 0.002 3 MKLTVNSSNS 0.002 V6-HLA-B7- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 2 EEIEFIVPKL 0.400 8 VPKLEHIEQD 0.200 7 IVPKLEHIEQ 0.050 5 EFIVPKLEHI 0.040 6 FIVPKLEHIE 0.010 10 KLEHIEQDER 0.003 3 EIEFIVPKLE 0.003 4 IEFIVPKLEH 0.002 1 SEEIEFIVPK 0.000 9 PKLEHIEQDE 0.000 V7-HLA-B7- l0 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 7 IVEDNISHEL 6.000 10 DNISHELFTL 4.000 18 TLHPEPPRWT 0.150 20 HPEPPRWTKK 0.060 5 HVIVEDNISH 0.050 3 DFHVIVEDNI 0.040 17 FTLHPEPPRW 0.020 6 VIVEDNISHE 0.010 15 ELFTLHPEPP 0.010 11 NISHELFTLH 0.010 12 ISHELFTLHP 0.010 9 EDNISHELFT 0.010 19 LHPEPPRWTK 0.002 2 HDFHVIVEDN 0.002 4 FHVIVEDNIS 0.002 16 LFTLHPEPPR 0.002 14 HELFTLHPEP 0.001 8 VEDNISHELF 0.001 13 SHELFTLHPE 0.000 1 THDFHVIVED 0.000 21 PEPPRWTKKP 0.000

TABLE XX V1-HLA- B3501-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 539 NPRIPKLHM 120.000 133 LPKEKIDPL 120.000 740 QPKEMIIKW 60.000 542 IPKLHMLEL 60.000 954 LPKKLNGNL 60.000 991 KPSWHLSNL 40.000 159 LPPLHIYWM 40.000 690 LPLAPFVRY 40.000 1172 QPTESADSL 40.000 130 VPKLPKEKI 24.000 299 KGRETKENY 24.000 1082 RGREYAGLY 24.000 47 FPFDEYFQI 24.000 197 DSRNDYCCF 22.500 151 LPCNPPKGL 20.000 1175 ESADSLVEY 20.000 390 HPFAGDVVF 20.000 2 EPLLLGRGL 20.000 988 TPSKPSWHL 20.000 154 NPPKGLPPL 20.000 768 KPQGAPVEW 20.000 84 IPSNNSGTF 20.000 316 VSYQDKGNY 15.000 285 TPQVDWNKI 12.000 398 FPREISFTN 12.000 250 KPKLLLPPT 12.000 69 WTKDGNPFY 12.000 210 RLRTIVQKM 12.000 111 ASNKLGIAM 10.000 886 FSGQRNSGM 10.000 917 GPESEPYIF 9.000 915 GAGPESEPY 9.000 310 TLKIENVSY 9.000 1127 YSVKEKEDL 7.500 384 GSPVDNHPF 7.500 1000 NATTKYKFY 6.000 1019 KPITEESST 6.000 810 GPDPQSVTL 6.000 597 VTLEDQGIY 6.000 1163 SLRSLNRDM 6.000 247 KQRKPKLLL 6.000 1214 GSSTATFPL 5.000 950 LSWGLPKKL 5.000 455 YSAFLHCEF 5.000 465 ASPEAVVSW 5.000 182 MSQKGDLYF 5.000 358 YSTGSNGIL 5.000 419 ASNVHGTIL 5.000 667 GNKEEPGRW 4.500 117 IAMSEEIEF 4.500 268 ILKGEILLL 4.500 1158 KPLKGSLRS 4.000 385 SPVDNHPFA 4.000 853 RVHGRLKGY 4.000 957 KLNGNLTGY 4.000 828 APVIHGVDV 4.000 352 KPQSAVYST 4.000 795 APYDVKVQA 4.000 157 KGLPPLHIY 4.000 772 APVEWEEET 4.000 629 DPPENLHLS 4.000 212 RTIVQKMPM 4.000 1104 CAIALLTLL 3.000 682 QGKKTTVIL 3.000 692 LAPFVRYQF 3.000 45 VAFPFDEYF 3.000 441 IQTKDGENY 3.000 1106 IALLTLLLL 3.000 6 LGRGLIVYL 3.000 1035 IGKISGVNL 3.000 857 RLKGYQINW 3.000 456 SAFLHCEFF 3.000 758 GPGLEYRVT 3.000 584 RIIIDGANL 3.000 245 SIKQRKPKL 3.000 838 NSTLVKVTW 2.500 726 APDRNPQNI 2.400 611 TALDSAADI 2.400 23 KAIEIPSSV 2.400 1152 YSDSDEKPL 2.250 5 LLGRGLIVY 2.000 997 SNLNATTKY 2.000 738 ASQPKEMII 2.000 722 TPPAAPDRN 2.000 181 YMSQKGDLY 2.000 657 NISEYIVEF 2.000 812 DPQSVTLYS 2.000 890 RNSGMVPSL 2.000 626 DVPDPPENL 2.000 39 KQSKVQVAF 2.000 44 QVAFPFDEY 2.000 730 NPQNIRVQA 2.000 283 LPTPQVDWN 2.000 508 YSCWVENAI 2.000 8 RGLIVYLMF 2.000 99 HISHFQGKY 2.000 736 VQASQPKEM 2.000 447 ENYATVVGY 2.000 755 EQNGPGLEY 2.000 1013 TSQGCGKPI 2.000 V2-HLA- B3501-9 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 9 FPKEKIDPL 120.000 6 VPKFPKEKI 24.000 1 FIVPSVPKF 1.000 3 VPSVPKFPK 0.200 5 SVPKFPKEK 0.010 2 IVPSVPKFP 0.010 4 PSVPKFPKE 0.005 8 KFPKEKIDP 0.003 7 PKFPKEKID 0.000 V2-B3501- 9 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 5 KGREAKENY 24.000 8 EAKENYGKT 1.800 3 LPKGREAKE 0.600 9 AKENYGKTL 0.030 1 GDLPKGREA 0.010 2 DLPKGREAK 0.010 7 REAKENYGK 0.003 4 PKGREAKEN 0.002 6 GREAKENYG 0.000 V2-B3501- 9 mers-(SET 3)-282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 2 SSTLGEGKY 10.000 5 LGEGKYAGL 0.300 6 GEGKYAGLY 0.200 3 STLGEGKYA 0.150 9 KYAGLYDDI 0.080 1 ESSTLGEGK 0.050 7 EGKYAGLYD 0.030 4 TLGEGKYAG 0.020 8 GKYAGLYDD 0.001 V3-B3501- 9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 33 CSKEQELSY 60.00 52 IQKSTSCNY 6.000 39 LSYRNRNML 5.000 10 TTYVSNTTY 2.000 24 GSPQPSIFI 2.000 25 SPQPSIFIC 2.000 38 ELSYRNRNM 2.000 31 FICSKEQEL 1.000 23 TGSPQPSIF 1.000 44 RNMLAEDFI 0.800 4 GVDVINTTY 0.600 56 TSCNYVEKS 0.500 13 VSNTTYVSN 0.500 22 ATGSPQPSI 0.400 60 YVEKSSTFF 0.300 21 NATGSPQPS 0.300 59 NYVEKSSTF 0.200 3 HGVDVINTT 0.200 27 QPSIFICSK 0.200 47 LAEDFIQKS 0.180 32 ICSKEQELS 0.150 58 CNYVEKSST 0.150 9 NTTYVSNTT 0.100 7 VINTTYVSN 0.100 54 KSTSCNYVE 0.100 15 NTTYVSNAT 0.100 8 INTTYVSNT 0.100 12 YVSNTTYVS 0.100 51 FIQKSTSCN 0.100 43 NRNMLAEDF 0.100 57 SCNYVEKSS 0.100 1 VIHGVDVIN 0.100 14 SNTTYVSNA 0.100 6 DVINTTYVS 0.100 42 RNRNMLAED 0.060 19 VSNATGSPQ 0.050 35 KEQELSYRN 0.040 62 EKSSTFFKI 0.040 46 MLAEDFIQK 0.030 40 SYRNRNMLA 0.030 5 VDVINTTYV 0.020 11 TYVSNTTYV 0.020 53 QKSTSCNYV 0.020 2 IHGVDVINT 0.015 45 NMLAEDFIQ 0.015 26 PQPSIFICS 0.010 49 EDFIQKSTS 0.010 16 TTYVSNATG 0.010 37 QELSYRNRN 0.010 55 STSCNYVEK 0.010 17 TYVSNATGS 0.010 29 SIFICSKEQ 0.010 18 YVSNATGSP 0.010 20 SNATGSPQP 0.010 50 DFIQKSTSC 0.010 28 PSIFICSKE 0.005 61 VEKSSTFFK 0.003 48 AEDFIQKST 0.003 36 EQELSYRNR 0.003 30 IFICSKEQE 0.001 41 YRNRNMLAE 0.001 34 SKEQELSYR 0.000 V4-HLA- B3501-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 8 DLPEQPTFL 2.000 1 VTLYSGEDL 1.000 7 EDLPEQPTF 0.150 4 YSGEDLPEQ 0.150 9 LPEQPTFLK 0.060 2 TLYSGEDLP 0.010 5 SGEDLPEQP 0.006 6 GEDLPEQPT 0.003 3 LYSGEDLPE 0.002 V5-HLA- B3501-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 4 LTVNSSNSI 0.400 3 KLTVNSSNS 0.200 8 SSNSIKQRK 0.050 7 NSSNSIKQR 0.050 1 PMKLTVNSS 0.030 9 SNSIKQRKP 0.010 6 VNSSNSIKQ 0.010 2 MKLTVNSSN 0.010 5 TVNSSNSIK 0.010 V6-HLA- B3501-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 7 VPKLEHIEQ 0.900 5 FIVPKLEHI 0.400 2 EIEFIVPKL 0.300 6 IVPKLEHIE 0.010 9 KLEHIEQDE 0.006 1 EEIEFIVPK 0.002 4 EFIVPKLEH 0.001 3 IEFIVPKLE 0.001 8 PKLEHIEQD 0.000 V7-HLA- B3501-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Start Subsequence Score 10 NISHELFTL 1.500 17 TLHPEPPRW 0.750 4 HVIVEDNIS 0.150 11 ISHELFTLH 0.100 8 EDNISHELF 0.100 9 DNISHELFT  0.100 19 HPEPPRWTK  0.060 3 FHVIVEDNI  0.040 5 VIVEDNISH  0.030 7 VEDNISHEL  0.030 18 LHPEPPRWT  0.020 2 DFHVIVEDN  0.010 16 FTLHPEPPR  0.010 14 ELFTLHPEP  0.010 6 IVEDNISHE  0.006 13 HELFTLHPE  0.001 1 HDFHVIVED 0.001 15 LFTLHPEPP 0.001 12 SHELFTLHP 0.000 20 PEPPRWTKK 0.000

TABLE XXI V1-HLA-B35- 10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 398 FPREISFTNL 120.000 849 VPKDRVHGRL 120.000 877 HPKEVNILRF 120.000 539 NPRIPKLHML 60.000 1019 KPITEESSTL 60.000 536 SPKNPRIPKL 60.000 1158 KPLKGSLRSL 40.000 94 IPNEGHISHF 40.000 480 KPLEGRRYHI 32.000 218 MPMKLTVNSL 20.000 1054 EPGAEHIVRL 20.000 895 VPSLDAFSEF 20.000 752 KSMEQNGPGL 20.000 568 WSKDGEAFEI 18.000 795 APYDVKVQAI 16.000 40 QSKVQVAFPF 15.000 1143 SVKDETFGEY 12.000 810 GPDPQSVTLY 12.000 499 RTTEEDAGSY 12.000 772 APVEWEEETV 12.000 996 LSNLNATTKY 10.000 758 GPGLEYRVTW 10.000 1162 GSLRSLNRDM 10.000 297 LPKGRETKEN 9.000 58 EAKGNPEPTF 9.000 478 EVKPLEGRRY 9.000 627 VPDPPENLHL 9.000 828 APVIHGVDVI 8.000 74 NPFYFTDHRI 8.000 1172 QPTESADSLV 8.000 2 EPLLLGRGLI 8.000 566 LSWSKDGEAF 7.500 67 FSWTKDGNPF 7.500 110 FASNKLGIAM 6.000 309 KTLKIENVSY 6.000 349 WTKKPQSAVY 6.000 69 WTKDGNPFYF 6.000 785 TLRVMTPAVY 6.000 6 LGRGLIVYLM 6.000 302 ETKENYGKTL 6.000 954 LPKKLNGNLT 6.000 1118 FVKRNRGGKY 6.000 745 IIKWEPLKSM 6.000 914 KGAGPESEPY 6.000 1121 ESNGSSTATF 5.000 244 NSIKQRKPKL 5.000 455 YSAFLHCEFF 5.000 557 DSHLKHSLKL 5.000 358 YSTGSNGILL 5.000 1093 ISTQGWFIGL 5.000 473 WQKVEEVKPL 4.500 1080 ETRGREYAGL 4.500 934 QPTFLKVIKV 4.000 735 RVQASQPKEM 4.000 1077 DVIETRGREY 4.000 991 KPSWHLSNLN 4.000 693 APFVRYQFRV 4.000 373 EPQPTIKWRV 4.000 34 VPTIIKQSKV 4.000 1048 HPIEVFEPGA 4.000 538 KNPRIPKLHM 4.000 790 TPAVYAPYDV 4.000 440 LIQTKDGENY 3.000 315 NVSYQDKGNY 3.000 449 YATVVGYSAF 3.000 916 AGPESEPYIF 3.000 516 IGKTAVTANL 3.000 1203 GSKEKGSVES 3.000 798 DVKVQAINQL 3.000 1000 NATTKYKFYL 3.000 1044 TQKTHPIEVF 3.000 418 EASNVHGTIL 3.000 596 NVTLEDQGIY 3.000 875 RTHPKEVNIL 3.000 857 RLKGYQINWW 3.000 1104 CAIALLTLLL 3.000 245 SIKQRKPKLL 3.000 725 AAPDRNPQNI 2.400 155 PPKGLPPLHI 2.400 21 FSKAIEIPSS 2.250 274 LLLECFAEGL 2.000 159 LPPLHIYWMN 2.000 957 KLNGNLTGYL 2.000 132 KLPKEKIDPL 2.000 43 VQVAFPFDEY 2.000 29 SSVQQVPTII 2.000 264 SSITILKGEI 2.000 988 TPSKPSWHLS 2.000 260 SGSESSITIL 2.000 689 ILPLAPFVRY 2.000 158 GLPPLHIYWM 2.000 780 TVTNHTLRVM 2.000 4 LLLGRGLIVY 2.000 960 GNLTGYLLQY 2.000 967 LQYQIINDTY 2.000 255 LPPTESGSES 2.000 730 NPQNIRVQAS 2.000 788 VMTPAVYAPY 2.000 8 RGLIVYLMFL 2.000 406 NLQPNHTAVY 2.000 V2-HLA- B3501-10 mers-(SET 1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 FPKEKIDPLE 1.200 7 VPKFPKEKID 0.600 6 SVPKFPKEKI 0.400 4 VPSVPKFPKE 0.200 9 KFPKEKIDPL 0.200 1 EFIVPSVPKF 0.100 3 IVPSVPKFPK 0.010 2 FIVPSVPKFP 0.010 5 PSVPKFPKEK 0.005 8 PKFPKEKIDP 0.000 V2-HLA- B3501-10 mers-(SET 2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 ESSTLGEGKY 10.000 4 TLGEGKYAGL 2.000 2 SSTLGEGKYA 0.750 5 LGEGKYAGLY 0.600 8 GKYAGLYDDI 0.040 7 EGKYAGLYDD 0.030 3 STLGEGKYAG 0.010 6 GEGKYAGLYD 0.001 V2-HLA- B3501-10 mers-(SET 3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 2 ESSTLGEGKY 10.000 5 TLGEGKYAGL 2.000 3 SSTLGEGKYA 0.750 6 LGEGKYAGLY 0.600 9 GKYAGLYDDI 0.040 8 EGKYAGLYDD 0.030 10 KYAGLYDDIS 0.020 4 STLGEGKYAG 0.010 7 GEGKYAGLYD 0.001 1 EESSTLGEGK 0.001 V3-HLA- B3501-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 43 RNRNMLAEDF 6.000 4 HGVDVINTTY 4.000 52 FIQKSTSCNY 2.000 26 SPQPSIFICS 2.000 33 ICSKEQELSY 2.000 10 NTTYVSNTTY 2.000 22 NATGSPQPSI 1.200 39 ELSYRNRNML 1.000 59 CNYVEKSSTF 1.000 23 ATGSPQPSIF 1.000 53 IQKSTSCNYV 0.600 14 VSNTTYVSNA 0.500 40 LSYRNRNMLA 0.500 57 TSCNYVEKSS 0.500 25 GSPQPSIFIC 0.500 34 CSKEQELSYR 0.450 24 TGSPQPSIFI 0.400 60 NYVEKSSTFF 0.200 28 QPSIFICSKE 0.200 11 TTYVSNTTYV 0.200 47 MLAEDFIQKS 0.200 38 QELSYRNRNM 0.200 48 LAEDFIQKST 0.180 32 FICSKEQELS 0.150 58 SCNYVEKSST 0.150 2 VIHGVDVINT 0.150 62 VEKSSTFFKI 0.120 9 INTTYVSNTT 0.100 31 IFICSKEQEL 0.100 7 DVINTTYVSN 0.100 13 YVSNTTYVSN 0.100 17 TTYVSNATGS 0.100 21 SNATGSPQPS 0.100 55 KSTSCNYVEK 0.100 56 STSCNYVEKS 0.100 8 VINTTYVSNT 0.100 15 SNTTYVSNAT 0.100 5 GVDVINTTYV 0.060 20 VSNATGSPQP 0.050 44 NRNMLAEDFI 0.040 45 RNMLAEDFIQ 0.030 37 EQELSYRNRN 0.030 46 NMLAEDFIQK 0.015 19 YVSNATGSPQ 0.010 51 DFIQKSTSCN 0.010 1 PVIHGVDVIN 0.010 30 SIFICSKEQE 0.010 16 NTTYVSNATG 0.010 6 VDVINTTYVS 0.010 12 TYVSNTTYVS 0.010 3 IHGVDVINTT 0.010 50 EDFIQKSTSC 0.010 29 PSIFICSKEQ 0.005 36 KEQELSYRNR 0.004 41 SYRNRNMLAE 0.003 49 AEDFIQKSTS 0.003 35 SKEQELSYRN 0.003 61 YVEKSSTFFK 0.003 54 QKSTSCNYVE 0.001 42 YRNRNMLAED  0.001 27 PQPSIFICSK  0.001 18 TYVSNATGSP 0.001 V4-HLA- B35-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 LPEQPTFLKV 1.200 1 SVTLYSGEDL 1.000 8 EDLPEQPTFL 0.100 5 YSGEDLPEQP 0.100 6 SGEDLPEQPT 0.060 7 GEDLPEQPTF 0.045 9 DLPEQPTFLK 0.020 3 TLYSGEDLPE 0.015 2 VTLYSGEDLP 0.010 4 LYSGEDLPEQ 0.002 V5-HLA- B35-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 1 MPMKLTVNSS 2.000 4 KLTVNSSNSI 0.800 8 NSSNSIKQRK 0.050 9 SSNSIKQRKP 0.050 2 PMKLTVNSSN 0.030 6 TVNSSNSIKQ 0.010 10 SNSIKQRKPK 0.010 3 MKLTVNSSNS 0.010 7 VNSSNSIKQR 0.010 5 LTVNSSNSIK 0.010 V6-HLA- B35-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 8 VPKLEHIEQD 0.600 2 EEIEFIVPKL 0.200 5 EFIVPKLEHI 0.040 7 IVPKLEHIEQ 0.015 6 FIVPKLEHIE 0.010 10 KLEHIEQDER 0.009 3 EIEFIVPKLE 0.003 4 IEFIVPKLEH 0.001 1 SEEIEFIVPK 0.000 9 PKLEHIEQDE 0.000 V7-HLA- B35-10 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 15; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. Start Subsequence Score 10 DNISHELFTL 1.500 17 FTLHPEPPRW 0.750 7 IVEDNISHEL 0.600 18 TLHPEPPRWT 0.100 12 ISHELFTLHP 0.100 20 HPEPPRWTKK 0.060 3 DFHVIVEDNI 0.040 8 VEDNISHELF 0.030 6 VIVEDNISHE 0.020 4 FHVIVEDNIS 0.015 5 HVIVEDNISH 0.015 15 ELFTLHPEPP 0.010 11 NISHELFTLH 0.010 2 HDFHVIVEDN 0.010 9 EDNISHELFT 0.010 19 LHPEPPRWTK 0.002 16 LFTLHPEPPR 0.001 14 HELFTLHPEP 0.001 13 SHELFTLHPE 0.000 1 THDFHVIVED 0.000 21 PEPPRWTKKP 0.000

TABLE XXII HLA-V1- A1-9 mers-282P1G3 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 500 TTEEDAGSY 31 1144 VKDETFGEY 29 1078 VIETRGREY 27 173 HIEQDERVY 26 69 WTKDGNPFY 24 755 EQNGPGLEY 24 961 NLTGYLLQY 24 5 LLGRGLIVY 23 579 GTEDGRIII 23 789 MTPAVYAPY 23 816 VTLYSGEDY 23 350 TKKPQSAVY 22 597 VTLEDQGIY 22 903 EFHLTVLAY 22 1154 DSDEKPLKG 22 78 FTDHRIIPS 21 145 EGDPIVLPC 21 120 SEEIEFIVP 20 157 KGLPPLHIY 20 181 YMSQKGDLY 20 236 STEIGSKAN 20 316 VSYQDKGNY 20 1192 SEDGSFIGA 20 44 QVAFPFDEY 19 690 LPLAPFVRY 19 915 GAGPESEPY 19 919 ESEPYIFQT 19 997 SNLNATTKY 19 1119 VKRNRGGKY 19 1175 ESADSLVEY 19 257 PTESGSESS 18 489 IYENGTLQI 18 586 IIDGANLTI 18 598 TLEDQGIYC 18 627 VPDPPENLH 18 811 PDPQSVTLY 18 975 TYEIGELND 18 1021 ITEESSTLG 18 1082 RGREYAGLY 18 1173 PTESADSLV 18 62 NPEPTFSWT 17 99 HISHFQGKY 17 143 VEEGDPIVL 17 310 TLKIENVSY 17 343 VEEPPRWTK 17 434 VVDVRPLIQ 17 476 VEEVKPLEG 17 479 VKPLEGRRY 17 636 LSERQNRSV 17 669 KEEPGRWEE 17 957 KLNGNLTGY 17 1052 VFEPGAEHI 17 1083 GREYAGLYD 17 1129 VKEKEDLHP 17 1191 FSEDGSFIG 17 194 EEKDSRNDY 16 270 KGEILLLEC 16 336 THDFHVIVE 16 371 EGEPQPTIK 16 393 AGDVVFPRE 16 407 LQPNHTAVY 16 441 IQTKDGENY 16 447 ENYATVVGY 16 630 PPENLHLSE 16 786 LRVMTPAVY 16 810 GPDPQSVTL 16 853 RVHGRLKGY 16 878 PKEVNILRF 16 901 FSEFHLTVL 16 944 DKDTATLSW 16 968 QYQIINDTY 16 1022 TEESSTLGE 16 1094 STQGWFIGL 16 1152 YSDSDEKPL 16 49 FDEYFQIEC 15 299 KGRETKENY 15 318 YQDKGNYRC 15 326 CTASNFLGT 15 359 STGSNGILL 15 466 SPEAVVSWQ 15 482 LEGRRYHIY 15 580 TEDGRIIID 15 653 DHNSNISEY 15 658 ISEYIVEFE 15 747 KWEPLKSME 15 932 PEQPTFLKV 15 972 INDTYEIGE 15 1000 NATTKYKFY 15 1183 YGEGDHGLF 15 1193 EDGSFIGAY 15 HLA- V2-(SET1)-A1 9 mers-(SET1)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 4 PSVPKFPKE 13 1 FIVPSVPKF 8 HLA- V2-(SET2)-A1 9 mers-(SET2)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 5 KGREAKENY 15 9 AKENYGKTL 13 6 GREAKENYG 10 HLA- V2-(SET3)-A1 9 mers-(SET3)- 282P1G3 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 2 SSTLGEGKY 25 6 GEGKYAGLY 18 HLA- V3-A1-9 mers- 282P1G3 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 33 CSKEQELSY 27 4 GVDVINTTY 26 10 TTYVSNTTY 22 52 IQKSTSCNY 15 HLA- V4-A1-9 mers- 282P1G3 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 9 LPEQPTFLK 13 5 SGEDLPEQP 12 6 GEDLPEQPT 11 1 VTLYSGEDL 8 3 LYSGEDLPE 7 4 YSGEDLPEQ 6 HLA-V5-A1- 9 mers- 282P1G3 Each peptide is a portion of SEQ ID NO: 11; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 6 VNSSNSIKQ 7 4 LTVNSSNSI 6 8 SSNSIKQRK 6 7 NSSNSIKQR 4 9 SNSIKQRKP 4 2 MKLTVNSSN 3 HLA- V6-A1-9 mers- 282P1G3 Each peptide is a portion of SEQ ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 score 2 EIEFIVPKL 13 9 KLEHIEQDE 11 4 EFIVPKLEH 7

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LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130267024A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1. A method of delivering a cytotoxic agent to a cell that expresses a 282P1G3 protein comprising the amino acid sequence set forth in SEQ ID NO:3, said method comprising: providing the cytotoxic agent conjugated to an antibody or antigen-binding fragment thereof that binds specifically to the protein; and exposing the cell to the antibody-agent or fragment-agent conjugate.
 2. The method of claim 1, wherein the antibody is a monoclonal antibody.
 3. The method of claim 2, wherein the monoclonal antibody is a humanized antibody.
 4. The method of claim 2, wherein the antigen-binding fragment is a Fab, F(ab□)2, or Fv fragment.
 5. The method of claim 3, wherein the antibody is a fully human antibody.
 6. The method of claim 3, wherein the antibody or antigen-binding fragment thereof comprises a murine antigen binding domain.
 7. The method of claim 3, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.
 8. The method of claim 1, wherein the cytotoxic agent is selected from the group consisting of radioactive isotopes, chemotherapeutic agents and toxins.
 9. The method of claim 8, wherein the radioactive isotope comprises 212Bi, 131I, 131In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, or a radioactive isotope of Lu.
 10. The method of claim 8, wherein the chemotherapeutic agent is selected from the group consisting of taxol, topotecan, adriamycin, actinomycin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, gelonin, and calicheamicin.
 11. The method of claim 8, wherein the toxin comprises ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, ethidium bromide, dihydroxy anthracin dione, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha sarcin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl 065, or cisplatin.
 12. The method of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a pharmaceutically acceptable carrier.
 13. The method of claim 1, wherein the cell is a cancer cell.
 14. The method of claim 1, wherein the cancer cell is from pancreas, ovary or lymph node.
 15. A method of inhibiting growth of a cell expressing a 282P1G3 protein comprising the amino acid sequence set forth in SEQ ID NO:3, comprising: exposing the cell to an effective amount of a cytotoxic agent conjugated to an antibody or antigen-binding fragment thereof that binds specifically to the protein of the cell, whereby the growth of the cell is inhibited.
 16. The method of claim 15, wherein the antibody is a monoclonal antibody.
 17. The method of claim 16, wherein the monoclonal antibody is a humanized antibody.
 18. The method of claim 16, wherein the antigen-binding fragment is a Fab, F(ab□)2, or Fv fragment.
 19. The method of claim 17, wherein the antibody is a fully human antibody.
 20. The method of claim 17, wherein the antibody or antigen-binding fragment thereof comprises a murine antigen binding domain.
 21. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.
 22. The method of claim 15, wherein the cytotoxic agent is selected from the group consisting of radioactive isotopes, chemotherapeutic agents and toxins.
 23. The method of claim 22, wherein the radioactive isotope comprises 212Bi, 131I, 131In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, or a radioactive isotope of Lu.
 24. The method of claim 22, wherein the chemotherapeutic agent is selected from the group consisting of taxol, topotecan, adriamycin, actinomycin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, gelonin, and calicheamicin.
 25. The method of claim 22, wherein the toxin comprises ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, ethidium bromide, dihydroxy anthracin dione, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha sarcin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl 065, or cisplatin.
 26. The method of claim 15, wherein the antibody or antigen-binding fragment thereof further comprises a pharmaceutically acceptable carrier.
 27. The method of claim 15, wherein the cell is a cancer cell.
 28. The method of claim 15, wherein the cancer cell is from pancreas, ovary or lymph node. 